Systems and methods for cellular reprogramming of a plant cell

ABSTRACT

Plant cell fate and development is altered by treating cells with cellular reprogramming factors. Embryogenesis inducing morphogenic developmental genes are used as cellular reprogramming factors, specifically comprising polypeptides or polynucleotides encoding gene products for generating doubled haploids or haploid plants from gametes. Maize microspores treated by contacting the isolated cells with an exogenous purified, recombinant embryogenesis inducing morphogenic developmental gene polypeptide results in embryogenesis. The gametes of a maize plant develop into embryoids when transformed with a genetic construct including regulatory elements and structural genes capable of acting in a cascading fashion to alter cellular fate of plant cells. Developmental morphogenic proteins expressed from a genetic construct are used for ex situ treatment methods and for in planta cellular reprogramming.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.application Ser. No. 16/755,432, filed Apr. 10, 2020, which in turnclaims the benefit of PCT Application No. PCT/US2018/055561, filed Oct.12, 2018, which in turn claims the benefit of U.S. ProvisionalApplication No. 62/572,007, filed Oct. 13, 2017, all of which areincorporated by reference herein in their entireties.

FIELD OF THE DISCLOSURE

The present disclosure relates to the field of plant molecular biology,more particularly to developing recombinant inbred lines.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically asan XML formatted sequence listing with a file named7488-US-PCN_Sequence_Listing_ST26 created on Mar. 21, 2023 and having asize of 83,000 bytes. The sequence listing contained in this XMLformatted document is part of the specification and is hereinincorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

Plant breeding programs identify new cultivars by screening numerousplants to identify individuals with desirable characteristics. Largenumbers of progeny from crosses are typically grown and evaluated,ideally across multiple years and environments, to select the plantswith the most desirable characteristics.

Typical breeding methods cross two parental plants and the filial 1hybrid (F₁ hybrid), is the first filial generation. Hybrid vigor in acommercial F₁ hybrid is observed when two parental strains, (typicallyinbreds), from different heterotic groups are intercrossed. Hybridvigor, the improved or increased function of any biological qualityresulting after combining the genetic contributions of its parents, isimportant to commercial maize seed production and commercial hybridperformance improvements require continued development of new inbredparental lines.

Maize inbred line development methods use maternal (gynogenic) doubledhaploid production, in which maternal haploid embryos are selectedfollowing the fertilization of the ear of a plant resultant from afirst-generation cross that has been fertilized with pollen from aso-called “haploid inducer” line. Pollination of a female flower withpollen of a haploid inducer strain results in elevated levels of ovulesthat contain only the haploid maternal genome, as opposed to inheritinga copy of both the maternal and paternal genome, thus, creating maternalhaploid embryos. Ovules within the female flower are the products ofmeiosis and each maternal ovule is a unique meiotically recombinedhaploid genome, thereby allowing immature maternal haploid embryos to beisolated and treated using in vitro tissue culture methods that includechromosome doubling treatments to rapidly enable generating maternaldoubled haploid recombinant populations. Many maize maternal haploidembryos resultant from fertilizing a target plant with pollen from amaize haploid inducer line fail to regenerate into a fertile, doubledhaploid plant and few, if any, in vitro tissue culture and plantletregeneration methods propagate multiple, fertile plants from one haploidembryo. Thus, there is a need for improving methods of producing doubledhaploid plants applicable to maternal gamete doubled haploids in maize.

Most maize inbreds are recalcitrant to microspore isolation, in vitrotissue culture and plantlet regeneration methods to create paternal(androgenic) gamete doubled haploids. Thus, there is a need for a methodof producing doubled haploid plants applicable to paternal gametedoubled haploids in maize.

Plant breeders would thus benefit from methods of developing apopulation of recombinant inbred lines that do not require extensivepollination control methods or the prolonged time required forpropagating self-fertilized lines into isogenic states.

SUMMARY OF THE DISCLOSURE

In an aspect, a method of generating a haploid plant embryo comprising(a) obtaining an embryogenic microspore by providing a plant microsporeto modulate microspore embryogenesis in the plant microspore, anembryogenesis modulation factor selected from the group consisting of(i) an embryogenesis inducing polypeptide; or (ii) an embryogenesisinducing compound; or (iii) a combination of (i) and (ii); and (b)producing the haploid plant embryo from the embryogenic microspore isprovided. In an aspect, the embryogenesis inducing polypeptide is notproduced by a stably integrated recombinant DNA construct in themicrospore. In an aspect, the embryogenesis inducing compound is akinase inhibitor selected fromN-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,anthra(1,9-cd)pyrazol-6(2H)-one:4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole,or N-benzyl-2-(pyrimidin-4-ylamino)-1,3-thiazole-4-carboxamide. In anaspect, the embryogenesis inducing compound is hemin. In an aspect, theembryogenesis inducing polypeptide is selected from the group consistingof (i) a WUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM) polypeptideor an Ovule Development Protein 2 (ODP2) polypeptide; (iii) a LEC1polypeptide; (iv) a combination of (i) and (ii); and (v) a combinationof (i) and (iii). In an aspect, the embryogenesis inducing polypeptidefurther comprises a cell penetrating peptide (CPP). In an aspect, theembryogenesis modulation factor is present in a tissue culture media. Inan aspect, method comprising co-culturing the microspore with anembryogenesis inducing suspension feeder cell culture, wherein theembryogenesis inducing suspension feeder cell culture expresses anembryogenesis inducing polypeptide or co-culturing the microspore withthe embryogenesis modulation factor in the culture media. In an aspect,the embryogenesis inducing polypeptide is selected from the groupconsisting of (i) a WUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM)polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; (iii)a LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) acombination of (i) and (iii). In an aspect, the method furthercomprising culturing the haploid plant embryo. In an aspect, the methodcomprising contacting the haploid plant embryo with a chromosomedoubling agent for a period sufficient to generate a doubled haploidplant embryo. In an aspect, the method wherein the microspore isobtained from maize, rice, sorghum, brassica, soybean, wheat, andcotton. In an aspect, the method wherein the embryogenesis modulationfactor comprises a cell penetrating peptide.

In an aspect, a method of generating a haploid plant embryo comprising(a) providing a plant comprising an expression cassette, wherein theexpression cassette comprises a tapetum cell preferred regulatoryelement operably linked to a polynucleotide encoding an embryogenesisinducing polypeptide; (b) crossing the plant of (a) with a wild typeinbred plant to provide an F₁ hybrid; (c) recovering an embryogenicmicrospore from the F₁ hybrid of (b); and (d) producing the haploidplant embryo from the embryogenic microspore is provided. In an aspect,the embryogenesis inducing polypeptide is a morphogenic developmentalpolypeptide. In an aspect, the morphogenic developmental polypeptide isselected from the group consisting of (i) a WUS/WOX homeoboxpolypeptide; (ii) a Babyboom (BBM) polypeptide or an Ovule DevelopmentProtein 2 (ODP2) polypeptide; (iii) a LEC1 polypeptide; (iv) acombination of (i) and (ii); and (v) a combination of (i) and (iii). Inan aspect, the method further comprising modifying genomic DNA by asite-specific nuclease. In an aspect, the expression cassette furthercomprises a polynucleotide encoding a site-specific nuclease. In anaspect, the site-specific nuclease is selected from the group consistingof a zinc finger nuclease, a meganuclease, TALEN, and a CRISPR-Casendonuclease. In an aspect, the CRISPR-Cas nuclease is Cas9 or Cpflnuclease. In an aspect, the modification of genomic DNA is made by a Casendonuclease during microspore embryogenesis. In an aspect, themodification of DNA is an insertion, a deletion, or a substitutionmutation. In an aspect, the Cas endonuclease is expressed from theexpression cassette, the Cas endonuclease further comprising a cellpenetrating peptide. In an aspect, the method further comprisingproviding a guide RNA expressed from the expression cassette. In anaspect, the modification of DNA is performed by providing a guide RNAand Cas endonuclease as a ribonucleoprotein complex exogenously to theembryogenic microspore. In an aspect, the plant is homozygous for theexpression cassette. In an aspect, the expression cassette furthercomprises a signal peptide. In an aspect, the expression cassettefurther comprises a cell penetrating peptide (CPP). In an aspect, themethod further comprising contacting the haploid plant embryo with achromosome doubling agent for a period sufficient to generate a doubledhaploid plant embryo. In an aspect, the plant is maize, rice, sorghum,brassica, soybean, wheat, or cotton. In an aspect, the method furthercomprising regenerating a doubled haploid plant from the doubled haploidplant embryo.

In an aspect, a method of generating a doubled haploid plant comprising(a) providing a plant comprising an expression cassette, wherein theexpression cassette comprises an endosperm cell preferred regulatoryelement operably linked to a polynucleotide encoding an embryogenesisinducing polypeptide; (b) crossing the plant of (a) with a wild type F₁hybrid; (c) recovering a haploid embryo from the cross of (b); (d)contacting the haploid embryo with a chromosome doubling agent for aperiod sufficient to generate a doubled haploid embryo; and (e)regenerating the doubled haploid plant from the doubled haploid embryoof (d) is provided. In an aspect, the embryogenesis inducing polypeptideis a morphogenic developmental polypeptide. In an aspect, themorphogenic developmental polypeptide is selected from the groupconsisting of (i) a WUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM)polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; (iii)a LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) acombination of (i) and (iii). In an aspect, the expression cassettefurther comprises a polynucleotide encoding a gene-editing nuclease. Inan aspect, the method further comprising modifying genomic DNA by asite-specific nuclease. In an aspect, the expression cassette furthercomprises a polynucleotide encoding a site-specific nuclease. In anaspect, the site-specific nuclease is selected from the group consistingof a zinc finger nuclease, a meganuclease, TALEN, and a CRISPR-Casendonuclease. In an aspect, the CRISPR-Cas nuclease is Cas9 or Cpflnuclease. In an aspect, the modification of genomic DNA is made by a Casendonuclease during haploid embryo embryogenesis. In an aspect, themodification of DNA is an insertion, deletion, or a substitutionmutation. In an aspect, the Cas endonuclease is expressed from theexpression cassette, the Cas endonuclease further comprising a cellpenetrating peptide. In an aspect, the method further comprisingproviding a guide RNA expressed from the expression cassette. In anaspect, the modification of DNA is performed by providing a guide RNAand Cas endonuclease as a ribonucleoprotein complex exogenously to theembryogenic haploid embryo. In an aspect, the plant is homozygous forthe expression cassette. In an aspect, the expression cassette furthercomprises a signal peptide. In an aspect, the expression cassettefurther comprises a cell penetrating peptide (CPP). In an aspect, theexpression cassette further comprises a polynucleotide encoding a colormarker or a fluorescent marker operably linked to regulatory element. Inan aspect, recovering the haploid embryo comprises screening for thepresence or the absence of the color marker, the fluorescent marker, orthe regulatory element. In an aspect, the screening occurs in a cellviability and cell sorting microfluidics device for automatedfluorescence detection for identifying, sorting, and selecting a haploidembryo comprising the expression cassette from a haploid embryo notcomprising the expression cassette.

In an aspect, an embryogenic microspore comprising an increased amountof an embryogenesis inducing polypeptide compared to a controlmicrospore, wherein the polypeptide is not produced in the microspore isprovided. In an aspect, an embryoid or embryogenic tissue produced fromthe embryogenic microspore is provided. In an aspect, an embryogenicmicrospore comprising a heterologous cellular reprogramming agent,wherein the heterologous cellular reprogramming agent is not produced inthe microspore is provided. In an aspect, the cellular reprogrammingagent is selected from the group consisting of (i) an embryogenesisinducing polypeptide; or (ii) an embryogenesis inducing compound; or(iii) a combination of (i) and (ii). In an aspect, the embryogenesisinducing polypeptide is selected from the group consisting of (i) aWUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM) polypeptide or anOvule Development Protein 2 (ODP2) polypeptide; (iii) a LEC1polypeptide; (iv) a combination of (i) and (ii); and (v) a combinationof (i) and (iii). In an aspect, the embryogenesis inducing compound ishemin or a kinase inhibitor or a combination thereof. In an aspect, theembryogenic microspore is capable of producing a haploid embryo. In anaspect, the embryogenic microspore is a maize embryogenic microspore. Inan aspect, the embryogenic microspore is from rice, sorghum, brassica,soybean, wheat, or cotton. In an aspect, a plant cell comprising anexpression cassette, wherein the expression cassette comprises a tapetumcell preferred regulatory element operably linked to a polynucleotideencoding an embryogenesis inducing polypeptide, and wherein theembryogenesis inducing polypeptide is capable of being secreted ortransported into a microspore is provided. In an aspect, theembryogenesis inducing polypeptide comprises a cell penetrating peptide.In an aspect, the embryogenesis inducing polypeptide is a morphogenicdevelopmental polypeptide selected from the group consisting of (i) aWUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM) polypeptide or anOvule Development Protein 2 (ODP2) polypeptide; (iii) a LEC1polypeptide; (iv) a combination of (i) and (ii); and (v) a combinationof (i) and (iii). In an aspect, a plant cell comprising an expressioncassette, wherein the expression cassette comprises an endosperm cellpreferred regulatory element operably linked to a polynucleotideencoding an embryogenesis inducing polypeptide and wherein theembryogenesis inducing polypeptide is produced in an endosperm cell, theembryo surrounding region (ESR), the Basal Endosperm Transfer Layer(BETL) or a combination thereof and capable of being secreted ortransported into an embryo cell is provided. In an aspect, a populationof plant cells comprising the plant cell and the embryo cell, whereinthe embryo cell comprises the secreted or transported embryogenesisinducing polypeptide is provided. In an aspect, the embryogenesisinducing polypeptide is a morphogenic developmental polypeptide selectedfrom the group consisting of (i) a WUS/WOX homeobox polypeptide; (ii) aBabyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2)polypeptide; (iii) a LEC1 polypeptide; (iv) a combination of (i) and(ii); and (v) a combination of (i) and (iii).

DESCRIPTION OF THE FIGURES

FIG. 1A shows a stereo microscope micrograph of proembryo development ofATCC40520 cells cultured for 21 days post isolation in a 9% sucroseinduction medium (control).

FIG. 1B shows a stereo microscope micrograph of proembryo development ofATCC40520 cells cultured for 21 days post isolation in a 9% sucroseinduction medium supplemented with hemin (1 μM final concentration).

FIG. 1C shows a bar graph of embryo-like structure expression response(relative mRNA level) of four (4) embryogenesis biomarker genes(1—GRMZM2G145440; 2—GRMZM2G057852; 3—GRMZM2G162184; and 4—GRMZM2G037368)and of four (4) pollen maturation biomarker genes (5—GRMZM2G177391;6—GRMZM2G176595; 7—GRMZM2G469689; and 8—GRMZM2G126196) at a 99%confidence level (p<0.01) of ATCC40520 cells after 8 days in vitrotissue culture in a 9% sucrose induction medium supplemented with 1 μMhemin.

FIG. 1D shows a stereo microscope micrograph of proembryo development ofcells derived from inbred EH microspores cultured for 21 days postisolation in a 9% sucrose induction medium (control).

FIG. 1E shows a stereo microscope micrograph of proembryo development ofcells derived from inbred EH microspores cultured for 21 days postisolation in a 9% sucrose induction medium supplemented with hemin (1 μMfinal concentration).

FIG. 1F shows a bar graph of microspore developmental responserepresenting the percent responsiveness of multicellular structures(MCS) and embryo-like structures (ELS) derived from inbred EHmicrospores cultured for 21 days post isolation in a 9% sucroseinduction medium (control) and in a 9% sucrose induction mediumsupplemented with hemin (1 μM final concentration).

FIG. 2A shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium withoutPD0325901(N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide)(negative control).

FIG. 2B shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith PD0325901 (0.19 mM final concentration).

FIG. 2C shows a stereo microscope micrograph of ATCC40520 cells 7cultured for days post isolation in a 9% sucrose induction mediumsupplemented with PD0325901 (0.39 mM final concentration).

FIG. 2D shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith PD0325901 (1.5 mM final concentration).

FIG. 2E shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium withoutSP600125 (anthra(1,9-cd)pyrazol-6(2H)-one) (negative control).

FIG. 2F shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith SP600125 (0.19 mM final concentration).

FIG. 2G shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith SP600125 (6.25 mM final concentration).

FIG. 2H shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith SP600125 (50 mM final concentration).

FIG. 21 shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium withoutSB203580(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole)(negative control).

FIG. 2J shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith SB203580 (0.78 mM final concentration).

FIG. 2K shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith SB203580 (3.22 mM final concentration).

FIG. 2L shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith SB203580 (50 mM final concentration).

FIG. 2M shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium withoutthiazovivin(N-benzyl-2-(pyrimidin-4-ylamino)-1,3-thiazole-4-carboxamide) (negativecontrol).

FIG. 2N shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith thiazovivin (0.39 mM final concentration).

FIG. 2O shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith thiazovivin (12.5 mM final concentration).

FIG. 2P shows a stereo microscope micrograph of ATCC40520 cells culturedfor 7 days post isolation in a 9% sucrose induction medium supplementedwith thiazovivin (50 mM final concentration).

FIG. 3A shows Coomassie blue staining using a 12% Bis-tris gel with theSeeBlue® Plus2 Pre-Stained Standard (Thermo Fisher Scientific catalog#LC5925) (lane 1) and purified recombinant ZmWUS2-hexa histidine-tagprotein samples, replicate 1 (lane 2) and replicate 2 (lane 3).

FIG. 3B shows a western blot analysis of the purified recombinantZmWUS2-hexa histidine-tag proteins described in FIG. 3A using a primaryanti-His monoclonal antibody and a secondary anti-mouse-HRP antibody(1:5,000) with recombinant ZmWUS2-hexa histidine-tag protein replicate 1(lane 1), recombinant ZmWUS2-hexa histidine-tag protein replicate 2(lane 2) and the SeeBlue® Plus2 Pre-Stained Standard (lane 3).

FIG. 3C shows wild type microspore embryogenesis without a recombinantZmWUS2-hexa histidine-tag protein treatment.

FIG. 3D shows biological activity for inducing cellular reprogramming toactivate microspore embryogenesis in wild type microspores treated witha purified recombinant ZmWUS2-hexa histidine-tag protein. Amicrospore-derived embryo with a developed radicle and root hairs wasobserved.

FIG. 4A shows a stereo microscope micrograph of microspore embryogenesisdevelopment without a purified recombinant WUSCHEL protein and without atransfection reagent treatment after 32 days of culture in a 4% sucroseinduction medium under dark conditions.

FIG. 4B shows a stereo microscope micrograph of microspore embryogenesisdevelopment with a purified recombinant WUSCHEL protein and without atransfection reagent treatment after 32 days of culture in a 4% sucroseinduction medium under dark conditions.

FIG. 4C shows a stereo microscope micrograph of microspore embryogenesisdevelopment without a purified recombinant WUSCHEL protein and with atransfection reagent treatment after 32 days of culture in a 4% sucroseinduction medium under dark conditions.

FIG. 4D shows a stereo microscope micrograph of microspore embryogenesisdevelopment with a purified recombinant WUSCHEL protein and atransfection reagent treatment after 32 days of culture in a 4% sucroseinduction medium under dark conditions.

FIG. 5A is a schematic diagram of a construct for creating stable maizemicrospore activator strains expressing a WUSCHEL-GFP fusion protein.

FIG. 5B is a schematic diagram of a construct for creating stable maizemicrospore activator strains expressing a WUSCHEL protein.

FIG. 5C is a schematic diagram of a construct for creating stable maizemicrospore activator strains expressing a WUSCHEL-GLUCOCORTICOIDRECEPTOR (GR) fusion protein.

FIG. 6A is a schematic diagram depicting a transformation methodschematic to create a T₀ microspore activator strain.

FIG. 6B shows a western blot of protein samples isolated from anther (A)and leaf (L) tissue using a custom polyclonal anti-WUSCHEL antibody(protein standard (lane 1), lanes of purified, recombinant WUS-GFPfusion protein (lanes, 2, 3, and 4), anther pools (pool 1, lanes 7 and8; pool 2 lanes 9 and 10; pool 3, lanes 12 and 13; pool 4, lanes 15 and16; and pool 5, lanes 18 and 19, leaf samples are shown in lanes 11, 14,17, and 20).

FIG. 6C shows a western blot of protein samples isolated from anther (A)and leaf (L) tissue using an anti-GFP antibody (protein standard (lane1), lanes of purified, recombinant WUS-GFP fusion protein (lanes, 2, 3,and 4), anther pools (pool 1, lanes 7 and 8; pool 2 lanes 9 and 10; pool3, lanes 12 and 13; pool 4, lanes 15 and 16; and pool 5, lanes 18 and19, leaf samples are shown in lanes 11, 14, 17, and 20).

FIG. 7 is a schematic diagram depicting a method for selecting wild typemicrospore-derived embryos from a hemizygous Ms44-WUS microsporeactivator hybrid cross.

FIG. 8A shows a bar graph representing increased levels of embryogenicresponsiveness of in vitro microspore cultures from a hemizygousMs44-WUS activator hybrid cross in response to in vivo WUS-GFP activity.

FIG. 8B shows images of a microspore-derived embryo-like structurebefore and after embryo regeneration resultant in a microspore-derivedhaploid plant.

FIG. 8C shows an ideogram depicting meiotic recombination breakpointsper maize chromosome one to ten (Chr; x axis) with inherited allelicpatterns (Parent1—black regions; Parent 2—gray regions; non-informative,monomorphic—light gray regions) positioned in respect to genetic mapposition (cM (centimorgan); y axis).

FIG. 9 is a schematic diagram depicting a method for selecting wild typemicrospore-derived embryos from a hemizygous Ms44-WUS microsporeactivator T₀ transgenic hybrid using an immature F₁ embryo explant fortransformation.

FIG. 10 is a schematic diagram showing a construct with three expressioncassettes useful for creating a stable maize endosperm activator strain.

FIG. 11 is a schematic diagram depicting a method for selecting wildtype F_(1:2) derived maternal haploids resultant from an induction crossusing a hemizygous endosperm activator line to improve maternal doubledhaploid production.

FIG. 12A is a bar graph showing embryos F_(1:2) resultant from a haploidinduction cross using a hemizygous haploid inducer line. The averagehaploid embryo size (millimeters (mm); y axis) were determined forCFP-minus and CFP-positive endosperm (wild type and morphologicaldevelopmental gene, “DevGene”, classes, respectively) for each haploidand diploid embryo, respectively.

FIG. 12B is a bar graph showing the percent of germinated haploidembryos (y axis) per haploid induction cross using transgenic lines withvarying copy number of the endosperm activator trait (x axis).

FIG. 13 is a schematic diagram of a construct useful for creating astable maize endosperm activator strain with gene editing activity.

FIG. 14 is a schematic diagram depicting a method for selecting wildtype F_(1:2) derived maternal haploids resultant from an induction crossusing a hemizygous endosperm activator line in combination with CAS9delivery from the endosperm to maternal haploid embryos to improvematernal doubled haploid production of gene-edited progeny.

DETAILED DESCRIPTION

The disclosures herein will be described more fully hereinafter withreference to the accompanying figures, in which some, but not allpossible aspects are shown. Indeed, disclosures may be embodied in manydifferent forms and should not be construed as limited to the aspectsset forth herein; rather, these aspects are provided so that thisdisclosure will satisfy applicable legal requirements.

Many modifications and other aspects disclosed herein will come to mindto one skilled in the art to which the disclosed methods andcompositions pertain having the benefit of the teachings presented inthe following descriptions and the associated figures. Therefore, it isto be understood that the disclosures are not to be limited to thespecific aspects disclosed and that modifications and other aspects areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting. As used in the specification and in the claims, the term“comprising” can include the aspect of “consisting of.” Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the disclosed methods and compositions belong. In thisspecification and in the claims which follow, reference will be made toa number of terms which shall be defined herein.

As used herein the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisdisclosure belongs unless clearly indicated otherwise.

All patents, publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this disclosure pertains. All patents, publications and patentapplications are herein incorporated by reference in the entirety to thesame extent as if each individual patent, publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety.

The oxidized form of iron protoporphyrin IX, hemin, is an essentialregulator of gene expression in mammalian cells, and promotes the growthof hematopoietic progenitor cells by acting as a nonprotein prostheticgroup forming part of or combined with proteins including respirationcytochromes, gas sensors, P450 enzymes (CYPs), catalases, peroxidases,nitric oxide synthases (NOS), guanyl cyclases, and even transcriptionalfactors (Tsiftsoglou et al., (2006) Pharmacol Ther. 111:327-45).Furthermore, in mammalian cells heme has been reported to act like asignaling ligand in cell respiration and metabolism, suggesting that inaddition to being a key regulator of gene expression hemin may be auseful co-factor alone or in combination with other treatments toimprove stress responses, adaptive processes, and even transcription ofgenes to prevent cell damage.

For plant cells and maize microspores in particular, methods ofimproving cellular reprogramming developmental fate toward embryogenesisinclude the need to improve stress adaptive processes caused by cellseparation and isolation techniques. Methods to inhibit proplastidswithin microspores from developing to amyloplast, or methods todedifferentiate an amyloplast to a proplastid, or to promote autophagywithin maize microspores are desirable.

Based on experiments in cultured cells, hemin blocks nuclear geneexpression. A regulatory system of nuclear gene expressions wasmodulated by a plastid signal during differentiation of plastids intoamyloplasts. A retrograde signaling from the plastid was blocked usingheme.

The disclosure provides efficient and effective methods of producingpopulations of recombinant inbred lines including, but not limited to,methods of initiating embryogenesis in plant cells to enable generatingdoubled haploid recombinant populations. The disclosure also providesmethods of enabling cellular reprogramming and embryogenic growthstimulation in non-transformed cells, and particularly in gametes orhaploid cells during the development of the gametes or haploid cells Thepresent disclosure provides methods of promoting microsporeembryogenesis in a cell, tissue or organ of a plant by contacting thecell, tissue or organ with an embryogenesis modulation factor capable ofreprogramming the cell, tissue or organ wherein embryogenesis is inducedin the cell, tissue or organ, such as, for example, an embryogenesisinducing exogenous morphogenic developmental gene protein product and/oran embryogenesis inducing compound. Embryogenesis inducing agents usefulin the methods of the disclosure include, but are not limited to proteinkinase inhibitor small molecules, such asN-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,anthra(1,9-cd)pyrazol-6(2H)-one:4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole,or N-benzyl-2-(pyrimidin-4-ylamino)-1,3-thiazole-4-carboxamide. Hemin isalso useful in the methods of the disclosure for inducing embryogenesis.

Also provided in many aspects are methods of generatingmicrospore-derived doubled haploid populations by ectopically expressingin a plant tissue or organ a fusion protein gene product ofembryogenesis inducing morphogenic developmental gene and atranslocation signal enabling cellular reprogramming and embryogenicgrowth stimulation in non-transformed cells, and particularly in gametesor haploid cells during the development of the gametes or haploid cells.In another aspect, the disclosure provides methods for generating in aplant tissue or organ microspore-derived doubled haploid populationusing embryogenesis inducing morphogenic developmental gene operablylinked to a translocation signal and a fluorescent protein and selectingbased on the presence or absence of the embryogenesis inducingmorphogenic developmental gene/translocation signal/fluorescent proteinfusion enabling cellular reprogramming and embryogenic growthstimulation in non-transformed cells, and particularly in gametes orhaploid cells during the development of the gametes or haploid cells.The disclosure provides in many aspects methods for reprogrammingmicrospores by co-culturing microspores with a purified protein, such asa morphogenic developmental embryogenesis inducing gene product with andwithout an embryogenesis inducing compound treatment. In another aspect,methods are provided for reprogramming microspores by co-culturingmicrospores in the presence of cells expressing a morphogenicdevelopmental embryogenesis inducing gene product. Periods ofco-cultivation (contact) with the embryogenesis inducing cellularreprograming agents will vary depending on the recalcitrance of themicrospores being treated. For example, in an aspect, microsporeembryogenesis is evidenced by the presence of multicellular structures(MCS) within the sporopollenin coat and/or rupturing of the exine of themicrospore and/or the presence of embryo-like structures (ELS). In anaspect, the microspores are co-cultured with the embryogenesis inducingcellular reprograming agents until certain characteristics such as MCSand/or ELS are observed. Alternatively, in an aspect other phenotypicand/or genotypic markers are also used to determine the embryogenicstate or the cellular reprogramming state. Generally, in many aspectsco-cultivation for periods of less than an hour, an hour, two hours,three hours, four hours, five hours, six hours, seven hours, eighthours, nine hours, ten hours, eleven hours, twelve hours, thirteenhours, fourteen hours, fifteen hours, sixteen hours, seventeen hours,eighteen hours, nineteen hours, twenty hours, twenty one hours, twentytwo hours, twenty three hours, twenty four hours, two days, three days,four days, five days, six days, seven days, eight days, nine days, tendays, eleven days, twelve days, thirteen days, fourteen days, fifteendays, sixteen days, seventeen days, eighteen days, nineteen days, twentydays, twenty one days, twenty two days, twenty three days, twenty fourdays, twenty five days, twenty six days, twenty seven days, twenty eightdays, twenty nine days, thirty days, thirty one days, thirty two days,thirty three days, thirty four days, thirty five days, thirty six days,thirty seven days, thirty eight days, thirty nine days, forty days,forty one days, forty two days, forty three days, forty four days, fortyfive days, forty six days, forty seven days, forty eight days, fortynine days, fifty days, fifty one days, fifty two days, fifty three days,fifty four days, fifty five days, fifty six days, fifty seven days,fifty eight days, fifty nine days, or sixty days or longer aresufficient for the cultured microspores to form MCS and/or ELS.Incubation or culturing period for inducing embryogenesis is optimizedbased on the type and the concentration of the embryogenesis inducingagent based on the guidance provided in this disclosure. The presentdisclosure also provides in many aspects methods of generatingmicrospore-derived doubled haploid populations, using the methodsdescribed above to promote microspore embryogenesis from a tissue ororgan of a filial plant resultant from a genetic cross of two differentstrains, such as a first generation F₁ hybrid or alternatively in laterfilial generations or back-cross generations, in a hemizygous transgeniccondition.

The present disclosure also provides in many aspects methods to promotemicrospore embryogenesis from a tissue or organ of a first generation F₁hybrid derived from transforming an F₁ embryo per se into said F₁ hybridregenerated directly in a hemizygous transgenic condition for thepurpose of generating a microspore-derived doubled haploid population.In a further aspect, the generated and/or treated microspores and/ormicrospore-derived cells are brought into contact with a chromosomedoubling agent to promote diploidization of the microspore-derivedembryoids. A further aspect of the disclosure provides methods forclonal propagation of plantlets derived from cells of a maternal haploidembryo produced by ectopic expression in a plant tissue or organ of amorphological developmental gene with or without a translocation signal.Also provided are in many aspects methods for clonal propagation ofmultiple gene edited plantlets derived from cells of a maternal haploidembryo produced by ectopic expression in a plant tissue or organ of amorphological developmental gene with or without a translocation signalfused to a gene product of a nuclease gene with or without atranslocation signal.

The disclosure also provides in maternally-derived haploid embryo cellsin many aspects methods of promoting embryogenesis in endosperm cellsand gene editing using a transformed haploid inducer line expressing anembryogenesis inducing gene product of a morphological developmentalgene with or without a translocation signal and a nuclease gene with orwithout a fertilization translocation signal. In a further aspect, thetreated maternal haploids embryos and/or embryo-derived cells arebrought into contact with a chromosome doubling agent to promotediploidization and regeneration of the maternally-derived somaticembryos.

As used herein, “reprogram” or “reprograming” or “reprogramed” is aprocess of reverting or sensitizing mature, specialized cells intoinduced pluripotent stem cells or into cells in an embryonic/embryogenicstate capable of being further developed into an embryo or embryo-likestructure. In a population of cells that are being “reprogrammed” notall cells are expected to be “reprogrammed” to the same extent or at thesame embryonic state. A mixture or mosaic nature of cells at variousstates of reprogramming is generally expected. Methods and compositionsprovided herein are expected to increase the ratio or percent of cellsthat are reprogrammed and in a desired embryogenic state compared tocells that have not been exposed to the methods and compositionsprovided herein. Reprograming also refers to the re-establishment ofgerm cell development. Reprograming can occur when an embryogenesisinducing polypeptide and/or a small molecule compound is contacted withplant cells rendering the plant cells embryogenic. In many aspects, themethods of the disclosure contact a haploid plant cell with anembryogenesis inducing agent such as for example, a polypeptide and/or asmall molecule compound to reprogram cell fate and cause the cell tobecome embryogenic. Alternatively, in many aspects a polynucleotideencoding an embryogenesis inducing polypeptide may be introduced andexpressed in a plant cell wherein the embryogenesis inducing polypeptideimpacts surrounding/adjacent cells thereby rendering the cellsembryogenic. The cells may be reprogrammed in planta or ex situ.Morphogenic (morphological) developmental genes and their embryogenesisinducing polypeptide products are useful in the disclosed methods. Asused herein, the term “morphogenic developmental gene” or “morphologicaldevelopmental gene” means a gene involved in plant embryogenesis,cellular reprograming, metabolism, organ development, stem celldevelopment, cell growth stimulation, organogenesis, regeneration,somatic embryogenesis initiation, accelerated somatic embryo maturation,initiation and/or development of the apical meristem, initiation and/ordevelopment of shoot meristem, initiation and/or development of shoots,or a combination thereof. Morphogenic developmental genes whenectopically expressed stimulate formation of a somatically-derivedstructure that can produce a plant. Ectopic expression of themorphogenic developmental gene stimulates the de novo formation of asomatic embryo or an organogenic structure, such as a shoot meristem,that can produce a plant. This stimulated de novo formation occurseither in the cell in which the morphogenic developmental gene isexpressed, or in a neighboring cell. A morphogenic developmental genecan be a transcription factor that regulates expression of other genes,or a gene that influences hormone levels in a plant tissue, both ofwhich can stimulate morphogenic changes. A morphogenic developmentalgene may be stably incorporated into the genome of a plant or it may betransiently expressed. Embryogenesis inducing morphogenic developmentalgenes include, but are not limited to WUS/WOX genes (WUS1, WUS2, WUS3,WOX2A, WOX4, WOX5, or WOX9) see U.S. Pat. Nos. 7,268,271, 7,309,813,7,348,468, 7,256,322, 7,994,391. 8,383,891, 8,581,037, and 9,029,641 andUnited States Patent Application Publications 20170121722, 20110258741,20100100981, 20040166563, and 20070271628, incorporated herein byreference in their entireties; Laux et al. (1996) Development 122:87-96;and Mayer et al. (1998) Cell 95:805-815; van der iGraaff et al., 2009,Genome Biology 10:248; Dolzblasz et al., 2016, Mol. Plant 19:1028-39.Modulation of WUS/WOX is expected to modulate plant and/or plant tissuephenotype including plant embryogenesis, cellular reprograming,metabolism, organ development, stem cell development, cell growthstimulation, organogenesis, regeneration, somatic embryogenesisinitiation, accelerated somatic embryo maturation, initiation and/ordevelopment of the apical meristem, initiation and/or development ofshoot meristem, initiation and/or development of shoots, or acombination thereof. Also of interest in this regard would be a MYB118gene (see U.S. Pat. No. 7,148,402), MYB 115 gene (see Wang et al. (2008)Cell Research 224-235), a BABYBOOM gene (BBM; see Boutilier et al.(2002) Plant Cell 14:1737-1749), an OVULE DEVELOPMENT PROTEIN 2 (ODP2)gene (see US20110010795, US20090328252, and US20050257289 incorporatedherein by reference in their entireties, or a CLAVATA gene (see, forexample, U.S. Pat. No. 7,179,963).

Other embryogenesis inducing morphogenic developmental genes suitablefor the present disclosure include, but are not limited to, LEC1 (Lotanet al., 1998, Cell 93:1195-1205), LEC2 (Stone et al., 2008, PNAS105:3151-3156; Belide et al., 2013, Plant Cell Tiss. Organ Cult113:543-553, and U.S. Pat. No. 8,865,971, incorporated herein byreference in its entirety), KN1/STM (Sinha et al., 1993. Genes Dev7:787-795), the IPT gene from Agrobacterium (Ebinuma and Komamine, 2001,In vitro Cell. Dev Biol-Plant 37:103-113), MONOPTEROS-DELTA (Ckurshumovaet al., 2014, New Phytol. 204:556-566), the Agrobacterium AV-6b gene(Wabiko and Minemura 1996, Plant Physiol. 112:939-951), the combinationof the Agrobacterium IAA-h and IAA-m genes (Endo et al., 2002, PlantCell Rep., 20:923-928), the Arabidopsis SERK gene (Hecht et al., 2001,Plant Physiol. 127:803-816), the Arabiopsis AGL15 gene (Harding et al.,2003, Plant Physiol. 133:653-663).

As used herein, the term “transcription factor” means a protein thatcontrols the rate of transcription of specific genes by binding to theDNA sequence of the promoter and either up-regulating or down-regulatingexpression. Examples of transcription factors, which may also serve asembryogenesis inducing morphogenic developmental genes, include membersof the AP2/EREBP family (including the BBM (ODP2), plethora andaintegumenta sub-families, CAAT-box binding proteins such as LEC1 andHAP3, and members of the MYB, bHLH, NAC, MADS, bZIP, RKD (US PatentApplication Publication No. 2013/0180010), and WRKY families.

The present disclosure in an aspect also includes plants obtained by anyof the disclosed methods or compositions herein. In many aspects, thepresent disclosure also includes seeds from a plant obtained by any ofthe disclosed methods or compositions herein. As used herein, the term“plant” refers to whole plants, plant organs (e.g., leaves, stems,roots, etc.), plant tissues, plant cells, plant parts, seeds,propagules, embryos and progeny of the same. As used herein, the termplant includes plant cells, plant protoplasts, plant cell tissuecultures from which plants can be regenerated, plant calli, plantclumps, and plant cells that are intact in plants or parts of plantssuch as embryos, pollen, ovules, seeds, leaves, flowers, branches,fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers,grain and the like. Plant cells include, without limitation, cells fromseeds, suspension cultures, explants, immature embryos, embryos, zygoticembryos, somatic embryos, embryogenic callus, meristem, somaticmeristems, organogenic callus, protoplasts, meristematic regions,embryos derived from mature ear-derived seed, leaf bases, leaves frommature plants, leaf tips, immature inflorescences, tassel, immature ear,silks, cotyledons, immature cotyledons, embryonic axes, meristematicregions, callus tissue, cells from leaves, cells from stems, cells fromroots, cells from shoots, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores. Plant cells can bedifferentiated or undifferentiated (e.g. callus, undifferentiatedcallus, immature and mature embryos, immature zygotic embryo, immaturecotyledon, embryonic axis, suspension culture cells, protoplasts, leaf,leaf cells, root cells, phloem cells and pollen). Plant parts includedifferentiated and undifferentiated tissues including, but not limitedto, roots, stems, shoots, leaves, pollen, seeds, tumor tissue andvarious forms of cells in culture (e. g., single cells, protoplasts,embryos, and callus tissue). The plant tissue may be in a plant or in aplant organ, tissue, or cell culture. Grain is intended to mean themature seed produced by commercial growers for purposes other thangrowing or reproducing the species. Progeny, variants and mutants of theregenerated plants are also included within the scope of the disclosure,provided these progeny, variants and mutants are made using the methodsand compositions disclosed herein and/or comprise the introducedpolynucleotides.

As used herein, the terms “transformed plant” and “transgenic plant”refer to a plant that comprises within its genome a heterologouspolynucleotide. Generally, the heterologous polynucleotide is stablyintegrated within the genome of a transgenic or transformed plant suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant DNA construct. It is to be understood that asused herein the term “transgenic” includes any cell, cell line, callus,tissue, plant part or plant the genotype of which has been altered bythe presence of a heterologous nucleic acid including those transgenicsinitially so altered as well as those created by sexual crosses orasexual propagation from the initial transgenic. A transgenic plant isdefined as a mature, fertile plant that contains a transgene.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct, including a nucleic acid expression cassettethat comprises a gene of interest, the regeneration of a population ofplants resulting from the insertion of the transferred gene into thegenome of the plant and selection of a plant characterized by insertioninto a particular genome location. An event is characterizedphenotypically by the expression of the inserted gene. At the geneticlevel, an event is part of the genetic makeup of a plant. The term“event” also refers to progeny produced by a sexual cross between thetransformant and another plant wherein the progeny include theheterologous DNA.

The compositions and methods of the present disclosure are applicable toa broad range of plant species, including dicotyledonous plants andmonocotyledonous plants. Representative examples of plants that can betreated in accordance with the methods disclosed herein include, but arenot limited to, wheat, cotton, sunflower, safflower, tobacco,Arabidopsis, barley, oats, rice, maize, triticale, sorghum, rye, millet,flax, sugarcane, banana, cassava, common bean, cowpea, tomato, potato,beet, grape, Eucalyptus, wheat grasses, turf grasses, alfalfa, clover,soybean, peanuts, citrus, papaya, Setaria sp, cacao, cucumber, apple,Capsicum, bamboo, melon, ornamentals including commercial garden andflower bulb species, fruit trees, vegetable species, Brassica species,as well as interspecies hybrids. In a preferred embodiment, thecompositions and methods of the disclosure are applied to maize plants.

The methods of the disclosure involve introducing a polypeptide,polynucleotide (i.e., DNA or RNA), or nucleotide construct (i.e., DNA orRNA) into a plant. As used herein, “introducing” means presenting to theplant the polynucleotide, polypeptide, or nucleotide construct in such amanner that the polynucleotide, polypeptide, or nucleotide constructgains access to the interior of a cell of the plant. The methods of thedisclosure do not depend on a particular method for introducing thepolynucleotide, polypeptide, or nucleotide construct into a plant, onlythat the polynucleotide, polypeptide, or nucleotide construct gainsaccess to the interior of at least one cell of the plant. Methods forintroducing polynucleotides, polypeptides, or nucleotide constructs intoplants are known in the art including, but not limited to, stabletransformation methods, transient transformation methods andvirus-mediated methods.

As used herein, a “stable transformation” is a transformation in whichthe polynucleotide or nucleotide construct introduced into a plantintegrates into the genome of the plant and is capable of beinginherited by the progeny thereof. “Transient transformation” means thata polynucleotide or nucleotide construct is introduced into the plantand does not integrate into the genome of the plant or a polypeptide isintroduced into a plant. In addition, “transient”, in certainembodiments may represent the presence of an embryogenesis inducingagent in a cell where such an agent has been exogenously applied orsecreted into from a neighboring cell or being produced from anextrachromosomal location (e.g., plasmid or another independentlyreplicating origin), or not produced by a stably integrated recombinantDNA construct within the same cell.

As used herein, “contacting”, “comes in contact with” or “in contactwith” are used to mean “direct contact” or “indirect contact” and meansthat the cells are place in a condition where the cells can come intocontact with any of the embryogenesis inducing substances disclosedherein including, but not limited to, an embryogenesis inducingmorphogenic developmental gene, a small molecule or a doubling agent.Such substance is allowed to be present in an environment where thecells survive (for example, medium or expressed in the cell or anadjacent cell) and can act on the cells. For example, the mediumcomprising a doubling agent may have direct contact with the haploidcell or the medium comprising the doubling agent may be separated fromthe haploid cell by filter paper, plant tissues, or other cells thus thedoubling agent is transferred through the filter paper or cells to thehaploid cell.

The methods provided herein rely upon the use of bacteria-mediatedand/or biolistic-mediated gene transfer to produce regenerable plantcells. Bacterial strains useful in the methods of the disclosureinclude, but are not limited to, a disarmed Agrobacteria, anOchrobactrum bacteria or a Rhizobiaceae bacteria. Standard protocols forparticle bombardment (Finer and McMullen, 1991, In Vitro Cell Dev.Biol.-Plant 27:175-182), Agrobacterium-mediated transformation (Jia etal., 2015, Int J. Mol. Sci. 16:18552-18543; US2017/0121722 incorporatedherein by reference in its entirety), or Ochrobactrum-mediatedtransformation (US2018/0216123 incorporated herein by reference in itsentirety) can be used with the methods and compositions of thedisclosure. Numerous methods for introducing heterologous genes intoplants are known and can be used to insert a polynucleotide into a planthost, including biological and physical plant transformation protocols.See, e.g., Miki et al., “Procedure for Introducing Foreign DNA intoPlants,” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson, eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Themethods chosen vary with the host plant and include chemicaltransfection methods such as calcium phosphate, microorganism-mediatedgene transfer such as Agrobacterium (Horsch, et al., (1985) Science227:1229-31), electroporation, micro-injection and biolisticbombardment. Expression cassettes and vectors and in vitro culturemethods for plant cell or tissue transformation and regeneration oftransgenic plants are known and available. See, e.g., Gruber, et al.,“Vectors for Plant Transformation,” in Methods in Plant MolecularBiology and Biotechnology, supra, pp. 89-119.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (Townsend, et al., U.S. Pat. No.5,563,055 and Zhao, et al., U.S. Pat. No. 5,981,840), direct genetransfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; 5,932,782; Tomes, et al., (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology6:923-926) and Lec1 transformation (WO 00/28058). See also, Weissinger,et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987)Particulate Science and Technology 5:27-37 (onion); Christou, et al.,(1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals ofBotany 75:407-413(rice); Ishida, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens), all of which are herein incorporated byreference in their entirety. Methods and compositions for rapid planttransformation are also found in U.S. 2017/0121722, herein incorporatedin its entirety by reference. Vectors useful in plant transformation arefound in U.S. patent application Ser. No. 15/765,521, hereinincorporated by reference in its entirety.

Methods for harvesting tassels, including sterilization methods, as wellas tassel pretreatments, for example, temperature pretreatments, areknown in the art and will vary depending on the intended tassel use.Specifically, prior to selecting tassels for microspore culture,microspores must be staged to an appropriate stage typically, betweenthe uninucleate to binucleate stage. Typically, for tassels with anthersand microspores at the appropriate stage, the tassels were detached andeach tassel is individually wrapped in for example, aluminum foil.

Isolation of microspores typically occurs after a tassel pretreatment ina reduced temperature environment to improve the androgenic response. Acommonly used technique is to place foil wrapped tassels at 10° C. forbetween 1 to 21 days. Additionally, preculture of anthers in a mannitolsolution, for example 0.3M liquid mannitol plus 50 mg/L ascorbic acid,can be practiced (U.S. Pat. Nos. 5,322,789 and 5,445,961 incorporatedherein by reference in their entireties).

Prior to use, tassels can be surface-sterilized in a 40% Clorox (8.25%Sodium Hypochlorite diluted v/v) solution plus two drops of Tween 80 forapproximately fifteen minutes, with gentle agitation on a reciprocalshaker. The tassels are then rinsed three or more times in sterile waterat room temperature and placed in a large petri dish and typically leftuncovered for 1-1.5 hours under aseptic conditions to allow any excesswater to evaporate. Another method known in the art includes placingspikelets detached from the tassel into permeable baskets that are thensubmerged in a 40% Clorox (8.25% Sodium Hypochlorite diluted v/v)solution plus two drops of Tween 80 for fifteen minutes followed byrinsing as described above. The spikelets are placed in a large petridish and typically left uncovered for 1-1.5 hours to allow excess waterto evaporate prior to microspore isolation.

A variety of isolation procedures for maize anthers and spikelets areknown in the art, including, but not limited to, glass rod macerationmethods (Pescitelli, et al., (1990) Plant Cell Rep. 8:628-31), blendingmethods, razor blade tissue cutting methods (see U.S. Pat. No. 5,445,961incorporated herein by reference in its entirety), tissue homogenizermethods (Gaillard, et al., (1991) Plant Cell Rep. 10:55-8), and tissuegrinder methods (Mandaron et al., (1990) Theor Appl Genet 80: 134-138.

Following isolation of microspores from the surrounding somatic tissue,the microspores are typically immediately after separating themicrospores from any anther debris placed into a fresh isolation medium.Numerous media compositions are known in the art. A common method ofseparating microspores from anther debris is to pass a blendedmicrospore anther debris slurry from the isolation procedure through asieve (Pescitelli (1989) Plant Cell Rep. 7:673-6, Gaillard, et al.,(1991), and U.S. Pat. No. 5,445,961 incorporated herein by reference inits entirety). Alternatively, the microspore anther debris slurry ispassed through several layers of cheesecloth or a mesh filter (Coumans,(1989) Plant Cell Rep. 7:618-21). Further separation can be performedusing a discontinuous density centrifugation method or additionalfiltration methods, including but not limited, to methods using asucrose or Percoll gradient (Coumans, (1989), Pescitelli et al.,(1990)). Alternatively, selection of cells captured at the 20-30%interface of a Percoll gradient ranging from 20-50% after centrifugationat 225 g for 3 min can be further separated using a final, high sucrose(0.44 M) centrifugation method (Gaillard, et al., (1991)). Furthervariations to separation methods are known in the art (Vergne et al.,(1991) In: Negrutiu I. (ed) BioMethods. Birkhauser, Basel, Boston,Bedinger and Edgerton, (1990) Plant Physiol. 92:474-9, Gaillard, et al.,(1991)) and can be optimized as needed.

Specific media used during isolation, for example, typically consists of6% sucrose, 50 mg/L acorbic acid, 400 mg/L proline, 0.05 mg/L biotin and10 mg/L nicotinic acid (see Petolino and Genovesi (1994) The MaizeHandbook, Freeling, M., Walbot, V. (eds) Springer-Verlag, New York).Various other media and solutions used for the culturing of maizemicrospores are similar to those used for other cereal tissue cultureprocedures and various modifications can be used (see Genovesi andMagill, (1982) Plant Cell Rep. 1:257-60, Martin and Widholm, (1996)Plant Cell Rep. 15:781-85, Magnard et al., (2000) Plant Mol Biol44:559-74, Testillano et al., (2002) Int J Dev Biol 46:1035-47,Testillano et al., (2004) Chromosoma 112:342-9, Shariatpanahi et al.,(2006) Plant Cell Rep 25:1294-9, Shim et al., (2006) Protoplasma228:79-86, Soriano et al., (2008) Plant Cell Rep 27:805-11, Cistue etal., (2009) Plant Cell Rep 28:727-35, Jacquard et al., (2009) Planta229:393-402, Jacquard et al., (2009) Plant Cell Rep 28:1329-39, Shim etal., (2009) Genome 52:166-74, Sanchez-Diaz et al., (2013) Plant Reprod26: 287-96). As evidenced in the citations above, common features formaize culture media typically include the use of N6, NLN, or YP basalsalt formulations with relatively high sugar concentrations (6-12%) thatmay have constituents including triiobenzoic acid, variousphytohormones, and/or proline.

The compositions and methods of the present disclosure include producingdoubled haploid plants from gametes by contacting a plant cell with amorphogenic developmental embryogenesis inducing gene protein productthat can induce cellular reprogramming and activate embryogenesis withinthe cell. An ex situ cellular reprogramming method for androgenicinduction by treating isolated microspores with a morphogenicdevelopmental embryogenesis inducing gene protein product, such as aWUSCHEL hexahistidine-tagged protein (“WUS-HISTAG”) (SEQ ID NO: 1 andSEQ ID NO: 2) is also provided. In another aspect, the presentdisclosure provides methods of treating isolated microspores with atranslational fusion protein comprising a morphogenic developmentalembryogenesis inducing gene protein product and a cell penetratingpeptide, more specifically a gamma-zein cell penetrating peptide (CPP)WUSCHEL hexahistidine-tagged translational fusion protein(“WUS-HISTAG-GZCPP”) (SEQ ID NO: 46 and SEQ ID NO: 47).

Also provided is an ex situ cellular reprogramming method for androgenicinduction by treating a plant cell with a morphogenic developmentalembryogenesis inducing gene protein product and/or an embryogenesisinducing small molecule compound, or combinations thereof, enablingimproved cellular reprogramming and embryogenic growth stimulation inwild type plant cells, including, but not limited to, gametic cells.

Methods of in planta cellular reprogramming for androgenic induction arealso provided by expressing a morphogenic developmental embryogenesisinducing gene protein product in a tissue-specific manner. Specifically,by expressing a morphogenic developmental embryogenesis inducing geneprotein product within tapetum cells of anthers, and more specificallyusing the Zea mays Ms44 promoter (“ZM-Ms44 PRO”; SEQ ID NO:3) and Ms44N-terminus secretion signal peptide (“Ms44^(SP)”; SEQ ID NO: 4 and SEQID NO: 5) fused to Zea mays WUSCHEL2 sequence (“ZM-WUS2”; SEQ ID NO: 6and SEQ ID NO: 7) to induce cellular reprogramming and activateembryogenesis within microspores.

In planta cellular reprogramming methods are also provided bytransforming a plant tissue or organ with a construct comprised of aWUSCHEL gene, a translocation signal, and linker sequence (“L3”; SEQ IDNO: 8 and SEQ ID NO: 9), which may also be fused to fluorescent proteingene, for example AC-GFP1 (SEQ ID NO: 10 and SEQ ID NO: 11), and aterminator sequence (“ZM-Ms44 TERM”; SEQ ID NO: 12) and then selectingwithin microspore-derived doubled haploid populations based on thepresence or absence of the transgene.

The disclosure also provides translational fusion proteins comprisingthe WUSCHEL polypeptide (SEQ ID NO: 7) and a translocation peptide or acellular localization signal sequence (SEQ ID NO: 13 (“WUS-virF^(C36)”),SEQ ID NO: 14 (“WUS-virF^(C36)”), SEQ ID NO: 15 (“WUS-virF^(C127)”), SEQID NO: 16 (“WUS-virF^(C127)”), SEQ ID NO: 17 (“WUS-GALLS (GS^(C27))”),SEQ ID NO: 18 (“WUS-GALLS (GS^(C27))”)) to create WUSCHEL variants foruse in the present methods as cellular reprogramming factors to induceembryogenesis in treated cells.

In certain aspects, the in planta cellular reprogramming methodsdisclosed herein also provide a construct comprised of a WUSCHEL geneand a glucocorticoid receptor (GR)-based fusion protein (“WUS-GR”; SEQID NO: 48 and SEQ ID NO: 49) to conditionally localize protein activityto the nucleus by external application of animal hormone analogs intothe in vitro tissue culture media.

The present disclosure also uses combinations of morphogenicdevelopmental genes and their embryogenesis inducing gene proteinproducts, such as a WUSCHEL protein and Z. mays ODP2 (ZM-ODP2; SEQ IDNO: 19 and SEQ ID NO: 20), an AP2/ERF transcription factor, or othermorphogenic developmental genes and their embryogenesis inducing geneprotein products known in the art. The present disclosure includes useof a translational fusion protein comprising a N-terminal Ms44N-terminus secretion signal peptide (ZM-Ms44^(SP); SEQ ID NO: 5) withthe Z. mays ODP2 polypeptide (ZM-ODP2; SEQ ID NO: 20) with a C-terminalcell penetrating peptide, including, but not limited to, the Z. maysknotted1 CPP (ZM-KNT1 CPP; SEQ ID NO: 21 and SEQ ID NO: 22), theSaccharomyces pombe TP10 CPP (SP-TP10 CPP; SEQ ID NO: 23 and SEQ ID NO:24), the Candida albicans Zebra CPP (CA-Zebra CPP; SEQ ID NO: 25 and SEQID NO: 26), the PEP1 CPP (PEP1 CPP; SEQ ID NO: 27 and SEQ ID NO: 28),the HIV-1 TAT CPP (HIV-1 TAT CPP; SEQ ID NO: 29 and SEQ ID NO: 30). Anysignal peptide or another moiety that is capable oftransporting/transferring/secreting the embryogenesis inducingpolypeptide into developing microspores or one or more of the embryocells in a maternal tissue is suitable for use with the compositionsdisclosed herein.

The present disclosure provides an ex situ cellular reprogramming methodfor androgenic induction by treating isolated microspores with atranslational fusion protein comprising an embryogenesis inducingmorphogenic developmental gene protein, such as a WUSCHELhexahistidine-tagged protein and C-terminal fusion using CPPs,including, but not limited to the CPP sequences described above.Androgenic induction can be obtained by treating isolated microsporeswith a translational fusion protein comprising an embryogenesis inducingmorphogenic developmental protein, such as a WUSCHEL protein andC-terminal fusion of a translocation signal, such as aWUSCHEL-virF^(C36) translational fusion, a WUSCHEL-virF^(C127)translational fusion, or a WUSCHEL-GALLS (GSC²⁷) translational fusionprotein.

Optionally, the ex situ methods of the present disclosure use isolatedmicrospores co-cultured with suspension “feeder cells” expressing anembryogenesis inducing morphogenic developmental polypeptide to furtherpromote cellular reprogramming to activate microspore embryogenesis.

Optionally, the ex situ cellular reprogramming methods of the presentdisclosure can be combined with and used with microspores isolated fromplant tissues generated using an in planta cellular reprogramming methoddisclosed herein.

The present disclosure provides an in planta cellular reprogrammingmethod for regenerating maternal haploid embryos by transforming a maizehaploid inducer line to stably integrate and express a heterologousexpression cassette encoding a morphological developmental polypeptidethat stimulates somatic embryogenesis and also encoding a secondcomponent including genes useful for gene editing purposes. Bothcomponents may comprise fusion peptides using secretion signal peptidesoperably linked to a promoter expressed within the endosperm. Secretionsignal peptides useful in the present disclosure include, but are notlimited to the Basal Endosperm Transfer Layer 9 (BETL9) secretion signalpeptide (“BETL9^(SP)”; SEQ ID NO: 31 and SEQ ID NO: 32) operably linkedto the BETL9 promoter (“ZM-BETL9 PRO”; SEQ ID NO: 33) or the BasalEndosperm Transfer Layer9-like (BETL9-like) secretion signal peptide(“BETL9-like^(SP)”; SEQ ID NO: 34 and SEQ ID NO: 35) operably linked tothe BETL9-like promoter (“ZM-BETL9-like PRO”; SEQ ID NO: 36). The inplanta cellular reprogramming methods may optionally use a fluorescentcolor marker expressed within the endosperm, for example thepolynucleotide encoding the Anemonia majano Cyan Fluorescent Protein(CFP) operably linked to the Zea mays FEM2 promoter (“AM-CFP-ZM-FEM2”;SEQ ID NO: 39).

Other reporter genes or selectable marker genes may also be included inthe expression cassettes of the present disclosure. Examples of suitablereporter genes known in the art can be found in, for example, Jefferson,et al., (1991) in Plant Molecular Biology Manual, ed. Gelvin, et al.,(Kluwer Academic Publishers), pp. 1-33; DeWet, et al., (1987) Mol. Cell.Biol. 7:725-737; Goff, et al., (1990) EMBO J. 9:2517-2522; Kain, et al.,(1995) Bio Techniques 19:650-655 and Chiu, et al., (1996) CurrentBiology 6:325-330, herein incorporated by reference in their entirety.

Selectable marker genes for selection of transformed cells or tissuescan include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate(Herrera Estrella, et al., (1983) Nature 303:209-213; Meijer, et al.,(1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron, et al., (1985)Plant Mol. Biol. 5:103-108 and Zhijian, et al., (1995) Plant Science108:219-227); streptomycin (Jones, et al., (1987) Mol. Gen. Genet.210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) TransgenicRes. 5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-36); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518), herein incorporated byreference in their entirety.

Other genes that could serve utility in the recovery of transgenicevents would include, but are not limited to, examples such as GUS(beta-glucuronidase; Jefferson, (1987) Plant Mol. Biol. Rep. 5:387), GFP(green fluorescence protein; Chalfie, et al., (1994) Science 263:802),luciferase (Riggs, et al., (1987) Nucleic Acids Res. 15(19):8115 andLuehrsen, et al., (1992) Methods Enzymol. 216:397-414) and the maizegenes encoding for anthocyanin production (Ludwig, et al., (1990)Science 247:449), herein incorporated by reference in their entirety.

The methods of the disclosure also provide for expression of multiplemorphological developmental genes in one expression cassette using apolycistronic linker (“TA-T2A”; SEQ ID NO: 40 and SEQ ID NO: 41)operably linked to a promoter and a second component including genesuseful for gene editing purposes, including, but not limited to, aStreptococcus pyogenes (CRISPR) CAS9 nuclease (“SP-CAS9”; SEQ ID NO: 42and SEQ ID NO: 43), or a Cpfl nuclease (“AC-Cpfl”; SEQ ID NO: 44 and SEQID NO: 45), or other nuclease proteins, including, but not limited to,zinc finger nucleases, meganucleases, or transcription activator-likeeffector nucleases. The use of the first component in a transformedmaize haploid inducer line for fertilizing the maternal ear of a targetplant is useful for improving doubled haploid production while thesecond component enables improving the regeneration of gene-edited,maize doubled haploids.

The present disclosure also provides methods of contacting haploid cellswith an amount of a chromosome doubling agent before, during, after, oroverlapping with any portion of the isolation and embryogenesisinduction process used for generating a paternal gamete (androgenic) ora maternal gamete (gynogenic) doubled haploid populations.

As used herein, the use of a cellular reprogramming agent (anembryogenesis inducing polypeptide or an embryogenesis inducingcompound) or a cellular reprogramming treatment of a plant cell outsideof the tissue of the organism, for example, extracted cells that havebeen isolated for experimentation and/or measurement done in an externalenvironment, is referred to as an “ex situ” treatment or treatmentmethod.

As used herein “recombinant” means a cell or vector, that has beenmodified by the introduction of a heterologous nucleic acid or a cellderived from a cell so modified. Thus, for example, a recombinant cellis a cell expressing a gene that is not found in identical form orlocation within the native (non-recombinant) cell or a cell thatexpresses a native gene in an expression pattern that is different fromthat of the native (non-recombinant) cell for example, the native geneis abnormally expressed, under expressed, has reduced expression or isnot expressed at all because of deliberate human intervention. The term“recombinant” as used herein does not encompass the alteration of a cellor vector by naturally occurring events (e.g., spontaneous mutation,natural transformation/transduction/transposition) such as thoseoccurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements, which permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed and apromoter.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

As used herein, the polypeptides useful in the methods of the disclosurecan be further engineered with a cell penetrating peptide, hereinreferred to as a “CPP”. CPPs useful in the present methods are a classof short peptides with a property to translocate across cell membranesand act as nanocarriers for protein delivery into plant cells. ExemplaryCPP families include, but are not limited to, CPPs derived from proteintransduction domains, amphipathic peptides, and synthetic cationicpolypeptides, such as polylysine, polyhistidine, and polyarginine, ordendrimeric polycationic molecules. Exemplary CPPs useful in the methodsof the disclosure include, but are not limited to, the peptide vascularendothelial-cadherin CPP, the transportan CPP, the monomer and dimer ofHIV-1 TAT basic domain Cpp, the penetratin CPP, synthetic cationichomoarginine oligopeptide CPPs (see Eudes and Chugh. (2008) Plant SignalBehav. 3:549-550) and the gamma zein CPP (see U.S. Pat. No. 8,581,036,incorporated herein by reference in its entirety). The presentdisclosure provides methods of using a gamma-zein CPP morphologicaldevelopmental protein translational fusion protein for use in contactingthe gamma-zein linked structure with a plant cell and allowing uptake ofthe gamma-zein linked structure into the plant cell to alter cell fateof the plant cell.

As used herein, a “cellular reprogramming factor” or an “embryogenesisinducing agent” includes, but is not limited to, small molecules,compounds, and morphological developmental embryogenesis inducing geneproducts that function in cell fate reprogramming either independentlyor in concert, including for example, microspore embryogenesisinduction. When a cell is contacted with a small molecule, it isbelieved that these reprogramming molecules activate expression ofendogenous genes within the cell eliciting an embryogenesis response inthe contacted cell. As used herein, a “cellular reprogramming treatment”is any of the treatments disclosed herein that elicits an embryogenesisresponse in the contacted cell.

As used herein, the use of a cellular reprogramming agent (anembryogenesis inducing polypeptide or an embryogenesis inducingcompound) or a cellular reprogramming treatment of a plant cell insideof the tissue of the organism, prior to cell isolation or cellextraction for experimentation and/or measurements done in an externalenvironment is referred to as an “in planta” treatment or treatmentmethod.

The term “regulatory element” refers to a nucleic acid molecule havinggene regulatory activity, i.e. one that has the ability to affect thetranscriptional and/or translational expression pattern of an operablylinked transcribable polynucleotide. The term “gene regulatory activity”thus refers to the ability to affect the expression of an operablylinked transcribable polynucleotide molecule by affecting thetranscription and/or translation of that operably linked transcribablepolynucleotide molecule. Gene regulatory activity may be positive and/ornegative and the effect may be characterized by its temporal, spatial,developmental, tissue, environmental, physiological, pathological, cellcycle, and/or chemically responsive qualities as well as by quantitativeor qualitative indications.

As used herein “promoter” is an exemplary regulatory element andgenerally refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequencecomprises proximal and more distal upstream elements, the latterelements are often referred to as enhancers. Accordingly, an “enhancer”is a nucleotide sequence that can stimulate promoter activity and may bean innate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different regulatory elements may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions.

A “plant promoter” is a promoter capable of initiating transcription inplant cells. Exemplary plant promoters include, but are not limited to,those that are obtained from plants, plant viruses and bacteria whichcomprise genes expressed in plant cells such as Agrobacterium orRhizobium. Examples are promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, seeds, fibers,xylem vessels, tracheids or sclerenchyma. Such promoters are referred toas “tissue preferred” promoters. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” or“regulatable” promoter is a promoter which is under environmentalcontrol. Examples of environmental conditions that may affecttranscription by inducible promoters include anaerobic conditions or thepresence of light. Another type of promoter is a developmentallyregulated promoter, for example, a promoter that drives expressionduring pollen development. Tissue preferred, cell type specific,developmentally regulated and inducible promoters are members of theclass of “non-constitutive” promoters. A “constitutive” promoter is apromoter that causes a nucleic acid fragment to be expressed in mostcell types at most times under most environmental conditions and statesof development or cell differentiation.

A “translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect numerous parameters including, processing of theprimary transcript to mRNA, mRNA stability and/or translationefficiency. Examples of translation leader sequences have been described(Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

As discussed above, one of skill will recognize the appropriate promoterto use to modulate paternal or maternal embryogenesis. For paternalembryogenesis, exemplary promoters include tassel-preferred promoters,anther-preferred promoters, and tapetum-preferred promoters. Knowntissue-specific, tissue-preferred or stage-specific regulatory elementsfurther include the anther-specific LAT52 (Twell, et al., (1989) Mol.Gen. Genet. 217:240-245), microspore-specific promoters such as the apggene promoter (Twell, et al., (1993) Sex. Plant Reprod. 6:217-224) andtapetum-specific promoters such as the TA29 gene promoter (Mariani, etal., (1990) Nature 347:737; U.S. Pat. No. 6,372,967), stamen-specificpromoters such as the MS26 gene promoter, MS44 gene promoter, MS45 genepromoter, the 5126 gene promoter, the BS7 gene promoter, the PG47 genepromoter (U.S. Pat. Nos. 5,412,085; 5,545,546; Zheng et al., (1993)Plant J 3(2):261-271), the SGB6 gene promoter (U.S. Pat. No. 5,470,359),G9 gene promoter (U.S. Pat. No. 5,8937,850; U.S. Pat. No. 5,589,610),the SB200 gene promoter (WO 2002/26789), and the like. Atissue-preferred promoter active in cells of male reproductive organs isparticularly useful in certain aspects of the present disclosure.

For maternal embryogenesis, exemplary promoters include seed-preferredpromoters. “Seed-preferred” promoters include both “seed-specific”promoters (those promoters active during seed development such aspromoters of seed storage proteins) as well as “seed-germinating”promoters (those promoters active during seed germination). See Thompsonet al. (1989) BioEssays 10:108, herein incorporated by reference. Suchseed-preferred promoters include, but are not limited to, the Cim1(cytokinin-induced message) promoter; the cZ19B1 (maize 19 kDa zein)promoter; and the milps (myo-inositol-1-phosphate synthase) promoter(see WO 00/11177 and U.S. Pat. No. 6,225,529 incorporated herein byreference in it entirety). Other promoters useful in the methods of thedisclosure include, but are not limited to, are endosperm-specificpromoters, such as the Gamma-zein promoter (Boronat et al. (1986) PlantScience 47:95-102) and embryo-specific promoters, such as the Globulin-1(Glob-1) promoter. For monocots, seed-specific promoters include, butare not limited to, the maize 15 kDa promoter, ther 22 kDa zeinpromoter, the 27 kDa zein promoter, the gamma-zein promoter, the waxypromoter, the shrunken 1 promoter, the shrunken 2 promoter, the globulin1 promoter, and the like. See also WO 00/12733, disclosingseed-preferred promoters from the end1 and end2 genes. Additionalseed-preferred promoters include the oleosin promoter (WO 00/0028058),the lipid transfer protein (LTP) promoter (U.S. Pat. No. 5,525,716), theLec promoter, the Jip1 promoter, and the milps3 promoter (see, WO02/42424).

As used herein, a “signal peptide” or “secretion signal peptide”sequence refers to a region of a protein interacting with a proteintransport system and translocates or targets a protein for delivery to aparticular destination. Examples of signal peptides or secretion signalpeptides useful in the methods of the disclosure include, but are notlimited to, signal-peptides targeting proteins to the extracellularmatrix of the plant cell, such as the Nicotiana plumbaginifoliaextension gene signal peptide (DeLoose, et al., (1991) Gene 99:95-100);signal peptides which cause proteins to be secreted, such as the PRIbsignal peptide (Lind, et al., (1992) Plant Mol. Biol. 18:47-53) or thebarley alpha amylase (BAA) signal peptide (Rahmatullah, et al., (1989)Plant Mol. Biol. 12:119).

Secretion signal peptides containing domains found in the superfamily ofbifunctional inhibitor/plant lipid transfer protein/seed storage helicaldomain proteins that characteristically encode eight conserved cysteineresidues important for secondary structure include, but are not limitedto, lipid transfer proteins such as LILY-LIM2 (Q43534), Sorghum(XP_002445754), Barley (BAK05897), Rice-OSC4 (BAD09233), Rice-MEN-8(XP_006660357) and Maize-MZm3-3 (NP_001105123) which are useful forengineering male-expressed plant-specific proteins useful in the methodsof the disclosure. Secretion signal-peptides targeting proteins from theendosperm to the embryo are useful for engineering female-expressedtranslational fusion proteins useful in the methods of the disclosure.

As used herein, “heterologous” refers to a nucleic acid that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention. For example, a promoter operably linkedto a heterologous structural gene that is from a species different fromthat from which the structural gene was derived, or, if from the samespecies, one or both are substantially modified from their original formand/or genomic location.

In an aspect, the embryogenesis inducing morphogenic developmental genesuseful in the methods of the disclosure can be provided in expressioncassettes for expression in the plant of interest. The cassette caninclude 5′ and 3′ regulatory sequences operably linked to anembryogenesis inducing morphogenic developmental gene sequence disclosedherein. “Operably linked” is intended to mean a functional linkagebetween two or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions (fusionproteins), by operably linked it is intended that the coding regions arein the same reading frame. The cassette may additionally contain atleast one additional gene to be co-transformed into the organism.Alternatively, the additional embryogenesis inducing morphogenicdevelopmental gene(s) can be provided on multiple expression cassettes.Such an expression cassette is provided with a plurality of restrictionsites for insertion of the embryogenesis inducing morphogenicdevelopmental gene sequence to be under the transcriptional regulationof the regulatory regions (promoter(s)). The expression cassette mayadditionally contain selectable marker genes.

As used herein, a chimeric signal peptide-morphogenic developmental genefusion can be further engineered with a translocation or a nuclearlocalization signal sequence on the C-terminus of the polypeptide topromote improved cellular reprogramming efficiency and embryogenesisinduction. The methods of the present disclosure provide a geneticconstruct encoding a WUSCHEL protein fused with a polypeptide derivedfrom bacterial virulence proteins conferring in planta translocation ofsecreted proteins. Agrobacterium tumefaciens and Agrobacteriumrhizogenes are examples of plant pathogens that can transferplasmid-encoded bacterial genes located on the transferred DNA (T-DNA)into plant cells in a manner dependent on the translocation of bacterialvirulence (Vir) proteins. Translocations of fusions between Crerecombinase with Vir protein polypeptides, specifically VirE2 or VirFpeptide sequences, directly demonstrated a role conferred by the Virpeptides for protein translocation into plant cells (Vergunst et al.,(2000) Science 290: 979-82). Further, the C-terminal 27 amino acids ofthe A. rhizogenes GALLS protein was shown to have a role in proteintransport and nuclear localization (Hodges et al., (2006) J. Bacteriol.188:8222-30). The use of peptides encoding translocation or nuclearlocalization signals are known in the art (see U.S. Pat. No. 6,800,791incorporated herein by reference in its entirety).

As used herein, expression cassettes useful in the methods of thedisclosure may contain a polynucleotide encoding a Ms44 signalpeptide-WUSCHEL fusion with a translocation or a nuclear localizationsignal sequence or a similar Ms44 signal peptide-ODP2 fusion with atranslocation fusion peptide which can be further engineered with a cellpenetrating peptide, herein referred to herein as a “CPP”. CPPs usefulin the present methods are a class of short peptides with a property totranslocate across cell membranes and act as nanocarriers for proteindelivery into plant cells. Exemplary CPP families include, but are notlimited to, CPPs derived from protein transduction domains, amphipathicpeptides, and synthetic cationic polypeptides, such as polylysine,polyhistidine, and polyarginine, or dendrimeric polycationic molecules.Exemplary CPPs useful in the methods of the disclosure include, but arenot limited to, the peptide vascular endothelial-cadherin CPP, thetransportan CPP, the monomer and dimer of HIV-1 TAT basic domain Cpp,the penetratin CPP, synthetic cationic homoarginine oligopeptide CPPs(see Eudes and Chugh. (2008) Plant Signal Behav. 3:549-550) and thegamma zein CPP (see U.S. Pat. No. 8,581,036, incorporated herein byreference in its entirety). The present disclosure provides methods ofusing a gamma-zein CPP morphological developmental protein translationalfusion protein for use in contacting the gamma-zein linked structurewith a plant cell and allowing uptake of the gamma-zein linked structureinto the plant cell to alter cell fate of the plant cell. Also providedfor use in the methods of the disclosure are engineered embryogenesisinducing morphogenic developmental proteins comprising a CPP fused tothe ODP2 protein for use in combination with a chimeric signalpeptide-WUSCHEL fusion protein. These genetic constructs are engineeredto deliver and contact a microspore with an embryogenesis inducingmorphogenic developmental protein comprising a CPP fused to the ODP2protein for use in combination with the chimeric signal peptide-WUSCHELfusion proteins operably linked to an anther-specific promoter, or morespecifically a tapetum-specific promoter.

As used herein, such genetic constructs can also be engineered todeliver and contact an embryo with an embryogenesis inducing morphogenicdevelopmental protein, more specifically a maize haploid embryo. Alsoprovided for use in the methods of the disclosure are expressioncassettes comprising a CPP fused to the ODP2 protein for use incombination with the chimeric signal peptide-WUSCHEL fusion proteinoperably whereby the proteins are engineered using genetic constructsdesigned with a chimeric endosperm or a transfer cell layer signalpeptide-WUSCHEL fusion protein operably linked to a endosperm-specificpromoter and polynucleotides encoding an endosperm or a transfer celllayer signal peptide-ODP2-CPP fusion peptide to translocate theexpressed proteins from the endosperm to the embryo.

As used herein, the “anther” is part of the stamen containing themicrosporangia that is attached to the filament. In angiosperms(flowering plants), the microsporangia produce microsporocyte, alsoknown as the microspore mother cell, which then produces fourmicrospores through meiosis. The microspores divide through mitosis tocreate pollen grains.

As used herein, the “locule” is a compartment within anthers containingthe male gametes during microgametogenesis.

The term “microgametogenesis” is the process in plant reproduction wherea microgametophyte, herein called a “microspore”, develops into atricellular pollen graint.

As used herein, the “microsporangium” or plural “microsporangia” is asporangium that produces spores that give rise to male gametophytes. Innearly all land plants, sporangia are the site of meiosis and producegenetically distinct haploid spores.

The term “microspore embryogenesis” means the activation of androgenicembryogenesis of microspores that results or induces microspores to bein an embryogenic state.

The term “microspore-derived embryo” or “microspore-derived embryoid”means a cell or cells derived from a microspore with a cell fate anddevelopment characteristic of cells undergoing embryogenesis.

The term “androgenic” means induction of androgenesis in which theembryo contains only paternal chromosomes (parthenogenesis) for haploidor diploid cells.

As used herein, a “haploid” plant has a single set (genome) ofchromosomes and the reduced number of chromosomes (n) in the haploidplant is equal to that in the gamete.

As used herein, a “diploid” plant has two sets (genomes) of chromosomesand the chromosome number (2n) is equal to that in the zygote.

As used herein, a “doubled haploid” or a “doubled haploid plant or cell”is one that is developed by the doubling of a haploid set ofchromosomes. A plant or seed that is obtained from a doubled haploidplant that is selfed any number of generations may still be identifiedas a doubled haploid plant. A doubled haploid plant is considered ahomozygous plant. A plant is a doubled haploid if it is fertile, even ifthe entire vegetative part of the plant does not consist of the cellswith the doubled set of chromosomes. For example, a plant will beconsidered a doubled haploid plant if it contains viable gametes, evenif it is chimeric.

As used herein, a “doubled haploid embryo” is an embryo that has one ormore cells containing 2 sets of homozygous chromosomes that can then begrown into a doubled haploid plant.

The term “medium” includes compounds in liquid, gas, or solid state.

The present disclosure provides methods in which the chromosomes may bedoubled at the microspore stage, at the embryo stage, at the mature seedstage, or anytime between pollination of the plant and before thegermination of the haploid seed. Alternatively, spontaneous doubling mayalso occur.

The ex situ methods of the present disclosure promote microsporeembryogenesis and cellular reprogramming by contacting an isolatedmicrospore with a embryogenesis inducing morphogenic developmentalprotein. Isolated microspores may be specifically contacted with anexogenous embryogenesis inducing morphogenic developmental protein toimprove maize microspore embryogenesis. For example, as disclosed hereinthe ex situ embryogenesis inducing morphogenic developmental proteintreatment cellular reprogramming method uses a heterologous expressionsystem to produce a purified, recombinant WUSCHEL protein (SEQ ID NO:2). The methods of the present disclosure include delivery of theprotein to the plant cell, for example using transfection reagents tofurther promote delivery of the exogenous WUSCHEL protein to theisolated microspore cells. In some aspects, the protein delivery method,with or without transfection reagents, can include electroporationmethods and/or sonication methods, performed in the presence of agentssuch as dimethyl sulfoxide (DMSO), adjuvants, surfactants, and the like,that further promote delivery of an exogenous embryogenesis inducingmorphogenic developmental protein into the microspore cells.

Also provided are, ex situ methods comprising contacting or treating anisolated microspore with an agent such as a small molecule or compoundthat enables cell fate reprogramming and stimulates embryogenic cellproliferation. The present disclosure provides methods comprisingco-culturing isolated microspores in an induction media supplementedwith a small molecule or compound. In some aspects, small-moleculeinhibitors of protein kinases are used in the methods of the disclosureto cellularly reprogram a plant cell.

The methods of the disclosure also provide combining the proteindelivery cellular reprogramming method, with or without transfectionreagents, with and without electroporation methods and/or sonicationmethods, which may be performed in the presence of agents such asdimethyl sulfoxide (DMSO), adjuvants, surfactants, and the likedescribed above and the cellular reprogramming treatments using a smallmolecule or compound described above to improve cellular reprogrammingof a plant cell.

The methods of the disclosure also provide that the ex situ and/or inplanta methods can subsequently include co-culturing the isolatedmicrospores in contact with maize suspension “feeder cells” possessingembryogenic and cellular reprogramming properties. In particular, themethod comprises co-culturing isolated microspores in the presence oftransgenic maize suspension cell cultures transformed with a geneticconstruct expressing an embryogenesis inducing morphogenic developmentalgene, such as the WUSCHEL protein (SEQ ID NO:7, and or ODP2 (SEQ IDNO:20).

In an aspect, the feeder cells are engineered to express polynucleotidesencoding polypeptides involved in growth stimulation, embryogenesis,cellular reprogramming, and/or cell cycle stimulation to increase thefrequency of haploid embryos, to increase the frequency of initiation ofmicrospore-derived embryos, and/or to stimulate and increase chromosomaldoubling efficiency. Polynucleotides useful in the methods of thedisclosure include, but are not limited to, embryogenesis inducingmorphogenic developmental genes and cell cycle genes including Cyclin A,Cyclin B, Cyclin C, Cyclin D, Cyclin E, Cyclin F, Cyclin G, and CyclinH; Pin1; E2F; Cdc25; RepA genes and similar plant viral polynucleotidesencoding replication-associated proteins. See U.S. Patent PublicationNo. 2002/0188965 incorporated herein by reference in its entirety.

In an aspect, the disclosure provides methods comprising co-culturingisolated microspores in the presence of non-transgenic maize suspensioncell cultures (feeder cells), more specifically using feeder cellsderived from genotypes with responsive androgenic phenotypes, such asfor example ATCC40520 or ATCC40519 (see U.S. Pat. No. 5,306,864 Aincorporated herein by reference in its entirety), or non-transgenic,responsive inbred strains such as HF1 (Martin and Widholm, (1996)).

The in planta method of the disclosure promotes embryogenesis from atissue or organ of a plant by ectopically expressing a morphologicaldevelopmental protein in a tissue or organ or in an adjacent tissue ororgan. Genetic elements providing spatiotemporal expression andlocalization to particular tissues or organs of a plant are useful inthe methods of the disclosure.

In an aspect, a promoter employed in the methods of the disclosure isthe native Z. mays Ms44 promoter (SEQ ID NO:3) resulting in exploitationof the spatiotemporal expression and localization characteristicproperties of Ms44, an anther-specific gene that is first detected inthe tapetum cells during meiosis that persists through uninucleatemicrospore development (see FIG. S1b in Fox et al., (2017) PlantBiotechnol. J., doi:10.1111/pbi.12689).

A signal peptide useful in the methods of the disclosure is the nativeZ. mays Ms44 signal peptide (SEQ ID NO:5; see also U.S. application Ser.Nos. 14/384,715, 14/384,743, 14/384,854 and 14/384,890 incorporatedherein by reference in their entireties).

In the present disclosure, a heterologous expression cassette encodingthe Ms44 promoter (SEQ ID NO: 3) regulating the anther-specificMs44signal peptide (SEQ ID NO: 5) is fused to a polynucleotide encodingthe WUSCHEL peptide (SEQ ID NO:7), thereby ectopically expressing theembryogenesis inducing morphogenic developmental gene duringmicrogametogenesis. The methods of the disclosure allow embryogenesisinducing morphogenic developmental gene protein synthesis and processingin the tapetum cells for secretion into the locule, thus resulting incontact with the microspores and activity of the embryogenesis inducingmorphogenic developmental protein to induce cellular reprogramming andactivate microspore embryogenesis.

As used herein, a “chimeric gene expression cassette” is an expressioncassette comprising a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence and caninclude in the 5′-3′ direction of transcription, a transcriptionalinitiation region (i.e., a promoter) and translational initiationregion, a secretion signal peptide, an embryogenesis inducingmorphogenic developmental gene sequence, a fluorescent protein sequence,and a transcriptional and translational termination region (i.e.,termination region) functional in plants.

In an aspect, genetic constructs useful in the methods of the disclosurein a polynucleotide encoding a Ms44 promoter and Ms44 secretion signalpeptide fused to a WUSCHEL protein which is also fused with a C-terminal36 amino acid VirF translocation peptide sequence (SEQ ID NO:14), hereincalled “virF^(c36)”, or is optionally fused to a C-terminal 127 aminoacid VirF translocation peptide sequence (SEQ ID NO:16), herein called“virF^(C127)”, or is is optionally fused to a 27 amino acidtranslocation signal peptide from the A. rhizogenes GALLS protein (SEQID NO:18), herein called “GS^(C27)” to promote increased morphogenicactivity and cellular reprogramming.

In an aspect, genetic constructs useful in the methods of the disclosurewith embryogenesis inducing morphogenic developmental gene proteinactivity (cellular reprogramming and embryogenesis induction activity)can also include fusion of the embryogenesis inducing morphogenicdevelopmental gene with a cell penetrating peptide to increase cellulardelivery and activity in a cell non-autonomous manner (increasing theembryogenesis inducing impact on surrounding/adjacent cells).

In an aspect, genetic constructs useful in the methods of the disclosurewith embryogenesis inducing morphogenic developmental gene proteinactivity (cellular reprogramming and embryogenesis induction activity)can also include fusion of the embryogenesis inducing morphogenicdevelopmental gene with a glucocorticoid receptor (GR)-based fusionprotein system (SEQ ID NO: 48 and SEQ ID NO: 49) to conditionallylocalize protein activity to the nucleus by external application ofanimal hormone analogs into the in vitro tissue culture.

Promoters useful in the methods of the disclosure include the ZmBETL9and 5′ untranslated region or ZmBETL9-like promoter and 5′ untranslatedregion (SEQ ID NO: 33 and SEQ ID NO:36, respectively) is fused to apolynucleotide encoding an embryogenesis inducing morphogenicdevelopmental gene, such as, the WUSCHEL peptide (SEQ ID NO:7) or theOVULE DEVELOPMENT PROTEIN 2 (ODP2) (SEQ ID NO: 20), thereby ectopicallyregulating embryogenesis inducing morphogenic developmental geneexpression during embryogenesis.

Endosperm secretion signal peptides, such as the N-terminal ZmBETL9secretion signal peptide or ZmBETL9-like secretion signal peptide (SEQID NO: 32 and SEQ ID NO: 35, respectively) which are fused to anembryogenesis inducing morphogenic developmental gene protein therebyenabling protein translocation from the endosperm to the embryo cellsduring embryogenesis are useful in the method of the disclosure.Optionally, a translational fusion protein comprising a secretion signalpeptide and an embryogenesis inducing morphogenic developmental geneprotein can be fused to a translocation signal peptide. In an aspect, atranslational fusion protein can comprise a cell penetrating peptide.The methods disclosed herein enable improved embryogenesis and cellularreprogramming in plant cells which also improve cellular responses insubsequent plant tissue culture methods.

The in planta cellular reprogramming methods of the disclosure improvematernal haploid embryo regeneration productivity and enable geneediting to provide regenerated gene-edited, maize doubled haploidswherein the treated cells, while not transgenic, are in contact with aembryogenesis inducing morphogenic developmental gene protein derivedfrom triploid endosperm cells comprising one paternal allele expressinga trait that is a stable transformant.

In some aspects, a heterologous expression cassette encoding the ZmBETL9promoter, 5′ untranslated region (SEQ ID NO 33), and the N-terminalZmBETL9 secretion signal peptide (SEQ ID NO: 31) or the ZmBETL9-likepromoter, 5′ untranslated region (SEQ ID NO: 36), and the N-terminalZmBETL9-like secretion signal peptide (SEQ ID NO: 34) is fused to apolynucleotide encoding an embryogenesis inducing morphogenicdevelopmental gene protein such as, the WUSCHEL peptide (SEQ ID NO:7) orthe OVULE DEVELOPMENT PROTEIN 2 (ODP2) peptide (SEQ ID NO: 20), is usedin the methods of the disclosure thereby ectopically regulatingembryogenesis inducing morphogenic developmental gene expression duringembryogenesis.

In an aspect, haploid cells can be contacted with an amount of achromosome doubling agent to promote chromosome doubling followed byregenerating homozygous diploid plants from the treated haploid cells.The haploid microspore cells can be in contact with the doubling agentbefore, during, or after initiation of microspore embryogenesis orembryo maturation. After chromosome doubling, the doubled haploid embryowill contain 2 copies of paternally derived chromosomes. The efficiencyof the process for obtaining doubled haploid plants from haploid embryosmay be greater than 10%, 20%, 30%, 50%, 60%, 70%, 80%, or 90%. Theduration of contact between the haploid cells and the chromosomaldoubling agent may vary. Contact may be from less than 24 hours, forexample 4-12 hours, to about a week. The duration of contact isgenerally from about 8 hours to 2 days.

Methods of chromosome doubling are disclosed in Antoine-Michard, S. etal., Plant cell, tissue organ cult., Cordrecht, the Netherlands, KluwerAcademic Publishers, 1997, 48(3):203-207; Kato, A., Maize GeneticsCooperation Newsletter 1997, 36-37; and Wan, Y. et al., TAG, 1989, 77:889-892. Wan, Y. et al., TAG, 1991, 81: 205-211. The disclosures ofwhich are incorporated herein by reference. Typical doubling methodsinvolve contacting the cells with colchicine, anti-microtubule agents oranti-microtubule herbicides, pronamide, nitrous oxide, or any mitoticinhibitor to create homozygous doubled haploid cells. The amount ofcolchicine used in medium is generally 0.01%-0.2% or approximately 0.05%of amiprophos-methyl (APM) (5-225 μM) may be used. The amount ofcolchicine can range from approximately 400-600 mg/L or approximately500 mg/L. The amount of pronamide in medium is approximately 0.5-20 μM.Examples of mitotic inhibitors are included in Table 1. Other agents maybe used with the mitotic inhibitors to improve doubling efficiency. Suchagents include dimethyl sulfoxide (DMSO), adjuvants, surfactants, andthe like.

TABLE 1 Common Name/Trade name CAS IUPAC Colchicine and ColchicineDerivatives colchicine/ (S)-N-(5,6,7,9-tetrahydro-1,2,3,10-acetyltrimethyl- tetramethoxy-9-oxobenzo (a) colchicinic acidheptalen-7-yl) acetamide colchicine derivatives Carbamates Carbetamide(R)-1-(ethylcarbamoyl)ethyl (2R)-N-ethyl-2- carbanilate[[(phenylamino)carbonyl]oxy]pro- panamide chloropropham ProphamBenzamides Pronamide/ 3,5-dichloro-N-(1,1-3,5-dichloro-N-(1,1-dimethyl-2- propyzamide dimethylpropynyl)benz-propynyl)benzamide amide Tebutam Benzoic Acids Chlorthal dimethyl(DCPA), Dicamba/dianat/ 3,6-dichloro-o-anisic acid3,6-dichloro-2-methoxybenzoic acid disugran (dicamba- methyl) (BANVEL,CLARITY) Dinitroaniline chromosome doubling agents benfluralin/benefin/N-butyl-N-ethyl-α,α,α- N-butyl-N-ethyl-2,6-dinitro-4- (BALAN)trifluoro-2,6-dinitro-p- (trifluoromethyl)benzenamine toluidine Butralin(RS)-N-sec-butyl-4-tert- 4-(1,1-dimethylethyl)-N-(1-butyl-2,6-dinitroaniline methylpropyl)-2,6- dinitrobenzenamine Chloralindinitramine N1,N1-diethyl-2,6-dinitro-4- N3,N3-diethyl-2,4-dinitro-6-trifluoromethyl-m- (trifluoromethyl)-1,3- phenylenediaminebenzenediamine ethalfluralin (Sonalan) N-ethyl-α,α,α-trifluoro-N-(2-N-ethyl-N-(2-methyl-2-propenyl)- methylallyl)-2,6-dinitro-p-2,6-dinitro-4- toluidine (trifluoromethyl)benzenamine fluchloralinN-(2-chloroethyl)-2,6- N-(2-chloroethyl)-2,6-dinitro-N-dinitro-N-propyl-4- propyl-4- (trifluoromethyl)aniline(trifluoromethyl)benzenamine or N-(2-chloroethyl)-α,α,α-trifluoro-2,6-dinitro-N- propyl-p-toluidine isopropalin4-isopropyl-2,6-dinitro-N,N- 4-(1-methylethyl)-2,6-dinitro-N,N-dipropylaniline dipropylbenzenamine methalpropalin α,α,α-trifluoro-N-(2-N-(2-methyl-2-propenyl)-2,6- methylallyl)-2,6-dinitro-N-dinitro-N-propyl-4- propyl-p-toluidine (trifluoromethyl)benzenaminenitralin 4-methylsulfonyl-2,6-dinitro-4-(methylsulfonyl)-2,6-dinitro-N,N- N,N-dipropylanilinedipropylbenzenamine oryzalin (SURFLAN) 3,5-dinitro-N4,N4-4-(dipropylamino)-3,5- dipropylsulfanilamide dinitrobenzenesulfonamidependimethalin N-(1-ethylpropyl)-2,6- N-(1-ethylpropyl)-3,4-dimethyl-2,6-(PROWL) dinitro-3,4-xylidine dinitrobenzenamine prodiamine5-dipropylamino-α,α,α- 2,4-dinitro-N3,N3-dipropyl-6-trifluoro-4,6-dinitro-o- (trifluoromethyl)-1,3- toluidine benzenediamineor 2,6-dinitro-N1,N1-dipropyl- 4-trifluoromethyl-m- phenylenediamineprofluralin N-cyclopropylmethyl-α,α,α-N-(cyclopropylmethyl)-2,6-dinitro- trifluoro-2,6-dinitro-N- N-propyl-4-propyl-p-toluidine (trifluoromethyl)benzenamine orN-cyclopropylmethyl-2,6- dinitro-N-propyl-4- trifluoromethylanilinetrifluralin (TREFLAN, α,α,α-trifluoro-2,6-dinitro-2,6-dinitro-N,N-dipropyl-4- TRIFIC, TRILLIN) N,N-dipropyl-p-toluidine(trifluoromethyl)benzenamine Phosphoroamidates APM (Amiprofos methyl);amiprophos- methyl Butamifos O-ethyl O-6-nitro-m-tolyl O-ethylO-(5-methyl-2-nitrophenyl) (RS)-sec- (1- butylphosphoramidothioatemethylpropyl)phosphoramidothioate Pyridines Dithiopyr Thiazopyr methyl2-difluoromethyl-5- methyl 2-(difluoromethyl)-5-(4,5-(4,5-dihydro-1,3-thiazol-2- dihydro-2-thiazolyl)-4-(2- yl)-4-isobutyl-6-methylpropyl)-6-(trifluoromethyl)-3- trifluoromethylnicotinatepyridinecarboxylate

The in planta methods of the disclosure provide stable transgenic“microspore activator” parental inbred lines useful in genetic crosseswith a second, wild type parent inbred line to create a first generationF₁ hybrid.

The methods of the disclosure, in an aspect, use this hemizygoustransgenic F₁ hybrid for generating an immature tassel that can produceflorets with anthers containing developing microspores. The microsporesare the products of meiosis, and thus, each male gamete has a uniquecombination of genes inherited from the parents along recombinedchromosomes due to chromosomal crossover events during meiosis. A singlecopy transgene that is at a single locus in a hemizygous state cansegregate in a 1:1 ratio during meiosis resulting in half of the gametesbeing wild type and the other half of the gametes having inherited thetransgenic locus. After meiosis, the wild type and transgenic gametescontinue to develop in planta with all developing microspores exposed tothe embryogenesis inducing morphogenic developmental gene protein whichis secreted from sporophytic tapetum cells originating from proteintranslation of the single copy of the transgene in the hemizygous F₁genome. Upon isolation of the microspores from the tassel tissues, themethods of the disclosure induce cellular programming activity duringmicrogametogenesis to improve microspore embryogenesis responsivenessand cellular reprograming in vitro. Selection of non-transgenicmicrospore-derived embryoids is performed using methods known to thoseskilled in the art.

In an aspect, two different inbred strains are cross-fertilized tocreate first generation F₁ zygotic embryos developing within thefertilized ear of the maternal parent. Each F₁ zygotic embryo has twosets (genomes) of chromosomes, one from each parent. The immature F₁zygotic embryos can be subsequently isolated from the maternal ear afterfertilization, for example 8 to 16 days after fertilization, fortransformation purposes to stably integrate into the F₁ plant genome apolynucleotide encoding an embryogenesis inducing morphogenicdevelopmental cellular reprogramming factor. In this manner, selectionof F₁ plants with a single copy of the embryogenesis inducingmorphogenic developmental cellular reprogramming genetic construct in ahemizygous state can be performed for sampling tassel tissues producingmicrospores within anthers. In respect to the inserted embryogenesisinducing morphogenic developmental cellular reprogramming transgene, themicrospores will segregate in a 1:1 ratio during gametogenesis resultingin half of the gametes being wild type and the other half of the gameteshaving inherited the transgenic embryogenesis inducing morphogenicdevelopmental cellular reprogramming locus. The methods of thedisclosure thereby allow for selecting F₂ generation wild-typemicrospores with improved embryogenesis responsiveness from a hemizygousF₁ hybrid for creating doubled haploid populations.

In an aspect, the methods of the disclosure also provide in plantaprotein delivery. The methods comprise transforming a maize haploidinducer line to stably integrate and express a heterologous expressioncassette, or cassettes, encoding two major functional activities: oneactivity comprising proteins for inducing somatic embryogenesis andcellular reprogramming and a second activity comprising proteins usefulfor gene editing purposes. Both components are operably linked to apromoter, or promoters, expressed within the endosperm, specifically theembryo surrounding region (ESR) and or the Basal Endosperm TransferLayer (BETL). The methods of the disclosure use the transformed haploidinducer line for fertilizing the maternal ear of a target plant togenerate haploid embryos with improved doubled haploid plantletregeneration and/or improved regeneration of gene-edited, doubledhaploid progeny. In these methods, expression of a heterologousexpression cassette comprising an embryogenesis inducing morphogenicdevelopmental gene protein from the paternal allele within triploidendosperm cells results in the proteins being translocated throughtransfer cells into the haploid embryo using secretion signal peptidescharacteristic of endosperm transfer cells. The present methods providematernal haploid embryo having increased levels of embryogenesis andplantlet regeneration capabilities once rescued haploid embryos arecultured in vitro.

A summary of SEQ ID NOS: 1-49 is presented in Table 2.

TABLE 2 Summary of SEQ ID NOS: 1-49, Polynucleotide (DNA) SEQ ID NO: orPolypeptide (PRT) Name Description 1 DNA WUS-histagWUS-hexahistidine-tagged coding sequence 2 PRT WUS-histagWUS-hexahistidine-tagged amino acid sequence 3 DNA ZM-Ms44 PRO Zea maysMs44 promoter sequence 4 DNA ZM-Ms44SP Zea mays Ms44 signal peptidecoding sequence 5 PRT ZM-Ms44SP Zea mays Ms44 signal peptide amino acidsequence 6 DNA ZM-WUS2 Zea mays WUS2 coding sequence 7 PRT ZM-WUS2 Zeamays WUS2 amino acid sequence 8 DNA L3 Linker3 coding sequence 9 PRT L3Linker3 amino acid sequence 10 DNA AC-GFP1 Aequorea coerulescens GFP1coding sequence 11 PRT AC-GFP1 Aequorea coerulescens GFP1 amino acidsequence 12 DNA ZM-Ms44 TERM Zea mays Ms44 terminator coding sequence 13DNA WUS-virFC36 WUS-virFC36 translational fusion coding sequence 14 PRTWUS-virFC36 WUS-virFC36 translational fusion amino acid sequence 15 DNAWUS-virFC127 WUS-virFC127 translational fusion coding sequence 16 PRTWUS-virFC127 WUS-virFC127 translational fusion amino acid sequence 17DNA WUS-GALLS WUS-GALLS (GSC27) translational (GSC27) fusion codingsequence 18 PRT WUS-GALLS WUS-GALLS (GSC27) translational (GSC27) fusionamino acid sequence 19 DNA ZM-ODP2 Zea mays ODP2 coding sequence 20 PRTZM-ODP2 Zea mays ODP2 amino acid sequence 21 DNA ZM-KNT1 CPP Zea maysknotted 1 CPP coding sequence 22 PRT ZM-KNT1 CPP Zea mays knotted 1 CPPamino acid sequence 23 DNA SP-TP10 CPP Saccharomyces pombe TP10 CPPcoding sequence 24 PRT SP-TP10 CPP Saccharomyces pombe TP10 CPP aminoacid sequence 25 DNA CA-Zebra CPP Candida albicans Zebra CPP codingsequence 26 PRT CA-Zebra CPP Candida albicans Zebra CPP amino acidsequence 27 DNA PEP1 CPP PEP1 CPP coding sequence 28 PRT PEP1 CPP PEP1CPP amino acid sequence 29 DNA HIV-1 TAT CPP HIV-1 TAT CPP codingsequence 30 PRT HIV-1 TAT CPP HIV-1 TAT CPP amino acid sequence 31 DNAZM-BETL9SP Zea mays Basal Endosperm Transfer Layer 9 secretion signalpeptide coding sequence 32 PRT ZM-BETL9SP Zea mays Basal EndospermTransfer Layer 9 secretion signal peptide amino acid sequence 33 DNAZM-BETL9 PRO Zea mays Basal Endosperm Transfer Layer 9 promoter codingsequence 34 DNA ZM-BETL9-likeSP Zea mays Basal Endosperm TransferLayer9-like secretion signal peptide coding sequence 35 PRTZM-BETL9-likeSP Zea mays Basal Endosperm Transfer Layer9-like secretionsignal peptide amino acid sequence 36 DNA ZM-BETL9-like Zea mays BasalEndosperm Transfer PRO Layer9-like promoter coding sequence 37 DNAODP2C445 ODP2C445-GALLSC27-FLAG coding sequence 38 PRT ODP2C445ODP2C445-GALLSC27-FLAG amino acid sequence 39 DNA AM-CFP-ZM- Anemoniamajano Cyan Fluorescent FEM2 Protein (CFP) operably linked to the Zeamays FEM2 promoter coding sequence 40 DNA TA-T2A Thosea asigna virus T2Acoding sequence 41 PRT TA-T2A Thosea asigna virus T2A amino acidsequence 42 DNA SP-CAS9 Streptococcus pyogenes (CRISPR) CAS9 nucleasecoding sequence 43 PRT SP-CAS9 Streptococcus pyogenes (CRISPR) CAS9nuclease amino acid sequence 44 DNA AC-Cpf1 MO Maize optimizedAcidaminococcus sp. strain BV3L6 Cpf1 nuclease coding sequence 45 PRTAC-Cpf1 Acidaminococcus sp. strain BV3L6 Cpf1 nuclease amino acidsequence 46 DNA WUS-histag- WUS-hexahistidine-tagged Gamma-zein GZCPPCPP translational fusion protein coding sequence 47 PRT GZCPP-WUS-WUS-hexahistidine-tagged Gamma-zein histag CPP translational fusionprotein amino acid sequence 48 DNA WUS-GR WUS glucocorticoid receptor(GR) fusion protein coding sequence 49 PRT WUS-GR WUS glucocorticoidreceptor (GR) fusion protein amino acid sequence

In an aspect, the disclosed methods and compositions can be used tointroduce into plant cells and organs with increased efficiency andspeed polynucleotides useful to target a specific site for modificationin the genome of a plant derived from the somatic embryo. Site specificmodifications that can be introduced with the disclosed methods andcompositions include those produced using any method for introducingsite specific modification, including, but not limited to, through theuse of gene repair oligonucleotides (e.g. US Publication 2013/0019349),or through the use of site-specific DNA cleaving technologies such asTALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like.For example, the disclosed methods and compositions can be used tointroduce a CRISPR-Cas system into a plant cell or plant, for thepurpose of genome modification of a target sequence in the genome of aplant or plant cell, for selecting plants, for deleting a base or asequence, for gene editing, and for inserting a polynucleotide ofinterest into the genome of a plant or plant cell. Thus, the disclosedmethods and compositions can be used together with a CRISPR-Cas systemto provide for an effective system for modifying or altering targetsites and nucleotides of interest within the genome of a plant, plantcell or seed. In an aspect, the Cas endonuclease gene is a plantoptimized Cas9 endonuclease, wherein the plant optimized Cas9endonuclease is capable of binding to and creating a double strand breakin a genomic target sequence the plant genome.

Genome-editing techniques such as zinc finger nucleases (ZFNs),transcription activator-like effector nucleases (TALENs), or homingmeganucleases, are available for producing targeted genomeperturbations.

The Cas endonuclease is guided by the guide nucleotide to recognize andoptionally introduce a double strand break at a specific target siteinto the genome of a cell. The CRISPR-Cas system provides for aneffective system for modifying target sites within the genome of aplant, plant cell or seed. Further provided are methods and compositionsemploying a guide polynucleotide/Cas endonuclease system to provide aneffective system for modifying target sites within the genome of a celland for editing a nucleotide sequence in the genome of a cell. Once agenomic target site is identified, a variety of methods can be employedto further modify the target sites such that they contain a variety ofpolynucleotides of interest. The disclosed compositions and methods canbe used to introduce a CRISPR-Cas system for editing a nucleotidesequence in the genome of a cell. The nucleotide sequence to be edited(the nucleotide sequence of interest) can be located within or outside atarget site that is recognized by a Cas endonuclease.

CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats)(also known as SPIDRs-SPacer Interspersed Direct Repeats) constitute afamily of recently described DNA loci. CRISPR loci consist of short andhighly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to140 times-also referred to as CRISPR-repeats) which are partiallypalindromic. The repeated sequences (usually specific to a species) areinterspaced by variable sequences of constant length (typically 20 to 58by depending on the CRISPR locus (WO2007/025097 published Mar. 1, 2007).

Cas gene includes a gene that is generally coupled, associated or closeto or in the vicinity of flanking CRISPR loci. The terms “Cas gene” and“CRISPR-associated (Cas) gene” are used interchangeably herein.

Cas endonuclease relates to a Cas protein encoded by a Cas gene, whereinthe Cas protein is capable of introducing a double strand break into aDNA target sequence. The Cas endonuclease is guided by the guidepolynucleotide to recognize and optionally introduce a double strandbreak at a specific target site into the genome of a cell. As usedherein, the term “guide polynucleotide/Cas endonuclease system” includesa complex of a Cas endonuclease and a guide polynucleotide that iscapable of introducing a double strand break into a DNA target sequence.The Cas endonuclease unwinds the DNA duplex in close proximity of thegenomic target site and cleaves both DNA strands upon recognition of atarget sequence by a guide nucleotide, but only if the correctprotospacer-adjacent motif (PAM) is approximately oriented at the 3′ endof the target sequence (see FIG. 2A and FIG. 2B of WO/2015/026883,published Feb. 26, 2015). In an aspect, the Cas endonuclease gene is aCas9 endonuclease.

In another aspect, the Cas endonuclease gene is plant, maize or soybeanoptimized Cas9 endonuclease, such as, but not limited to those shown inFIG. 1A of US2016/0208272, and incorporated herein by reference.

The term “Cas protein” or “Cas endonuclease” or “Cas nuclease” or “Caspolupeptide” refers to a polypeptide encoded by a Cas(CRISPR-associated) gene. A Cas protein includes but is not limited toCas9 protein, Cas9 orthologs, a Cpfl (Cas12) protein, a C2c1 protein, aC2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Cas10,or combinations or complexes of these. A Cas protein may be a “Casendonuclease”, that when in complex with a suitable polynucleotidecomponent, is capable of recognizing, binding to, and optionally nickingor cleaving all or part of a specific polynucleotide target sequence. ACas endonuclease described herein comprises one or more nucleasedomains. A Cas protein is further defined as a functional fragment orfunctional variant of a native Cas protein, or a protein that shares atleast 50%, between 50% and 55%, at least 55%, between 55% and 60%, atleast 60%, between 60% and 65%, at least 65%, between 65% and 70%, atleast 70%, between 70% and 75%, at least 75%, between 75% and 80%, atleast 80%, between 80% and 85%, at least 85%, between 85% and 90%, atleast 90%, between 90% and 95%, at least 95%, between 95% and 96%, atleast 96%, between 96% and 97%, at least 97%, between 97% and 98%, atleast 98%, between 98% and 99%, at least 99%, between 99% and 100%, or100% sequence identity with at least 50, between 50 and 100, at least100, between 100 and 150, at least 150, between 150 and 200, at least200, between 200 and 250, at least 250, between 250 and 300, at least300, between 300 and 350, at least 350, between 350 and 400, at least400, between 400 and 450, at least 500, or greater than 500 contiguousamino acids of a native Cas protein, and retains at least partialactivity.

The terms “guide RNA/Cas endonuclease complex”, “guide RNA/Casendonuclease system”, “guide RNA/Cas complex”, “guide RNA/Cas system”,“gRNA/Cas complex”, “gRNA/Cas system”, “RNA-guided endonuclease”, and“RGEN” are used interchangeably herein and refer to at least one RNAcomponent and at least one Cas endonuclease that are capable of forminga complex, wherein said guide RNA/Cas endonuclease complex can directthe Cas endonuclease to a DNA target site, enabling the Cas endonucleaseto recognize, bind to, and optionally nick or cleave (introduce a singleor double-strand break) the DNA target site. In some aspects, thecomponents are provided as a ribonucleoprotein complex (“RNP”) of a Casendonuclease protein and a guide RNA.

Described herein are methods for genome editing with CRISPR Associated(Cas) endonucleases during microspore embryogenesis or for portions ofthe microspore embryogenesis induction. Following characterization ofthe guide RNA (or guide polynucleotide) and PAM sequence, aribonucleoprotein (RNP) complex comprising the Cas endonuclease and theguide RNA (or guide polynucleotide) may be utilized to modify a targetpolynucleotide, including but not limited to: synthetic DNA, isolatedgenomic DNA, or chromosomal DNA in other organisms, including plants. Tofacilitate optimal expression and nuclear localization (for eukaryoticcells), the gene comprising the Cas endonculease may be optimized asdescribed in WO2016186953 published 24 Nov. 2016, and then deliveredinto cells as DNA expression cassettes by methods known in the art. Thecomponents necessary to comprise an active RNP may also be delivered asRNA with or without modifications that protect the RNA from degradationor as mRNA capped or uncapped (Zhang, Y. et al., 2016, Nat. Commun.7:12617) or Cas protein guide polynucleotide complexes (WO2017070032published 27 Apr. 2017), or any combination thereof. Additionally, apart or part(s) of the complex may be expressed from a DNA constructwhile other components are delivered as RNA with or withoutmodifications that protect the RNA from degradation or as mRNA capped oruncapped (Zhang et al. 2016 Nat. Commun. 7:12617) or Cas protein guidepolynucleotide complexes (WO2017070032 published 27 Apr. 2017) or anycombination thereof.

As related to the Cas endonuclease, the terms “functional fragment,”“fragment that is functionally equivalent,” and “functionally equivalentfragment” are used interchangeably herein. These terms refer to aportion or subsequence of the Cas endonuclease sequence of the presentdisclosure in which the ability to create a double-strand break isretained.

As related to the Cas endonuclease, the terms “functional variant,”“variant that is functionally equivalent” and “functionally equivalentvariant” are used interchangeably herein. These terms refer to a variantof the Cas endonuclease of the present disclosure in which the abilityto create a double-strand break is retained. Fragments and variants canbe obtained via methods such as site-directed mutagenesis and syntheticconstruction.

In addition to the double-strand break inducing agents, site-specificbase conversions can also be achieved to engineer one or more nucleotidechanges to create one or more edits into the genome. These include forexample, a site-specific base edit mediated by an C•G to T•A or an A•Tto G•C base editing deaminase enzymes (Gaudelli et al., Programmablebase editing of A•T to G•C in genomic DNA without DNA cleavage.” Nature(2017); Nishida et al. “Targeted nucleotide editing using hybridprokaryotic and vertebrate adaptive immune systems.” Science 353 (6305)(2016); Komor et al. “Programmable editing of a target base in genomicDNA without double-stranded DNA cleavage.” Nature 533 (7603)(2016):420-4. A catalytically “dead” or inactive Cas9 (dCas9), forexample a catalytically inactive “dead” version of a Cas9 orthologdisclosed herein, fused to a cytidine deaminase or an adenine deaminaseprotein becomes a specific base editor that can alter DNA bases withoutinducing a DNA break. Base editors convert C->T (or G->A on the oppositestrand) or an adenine base editor that would convert adenine to inosine,resulting in an A->G change within an editing window specified by thegRNA.

As used herein, the term “guide nucleotide” relates to a syntheticfusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variabletargeting domain, and a tracrRNA. In an aspect, the guide nucleotidecomprises a variable targeting domain of 12 to 30 nucleotide sequencesand a RNA fragment that can interact with a Cas endonuclease.

As used herein, the term “guide polynucleotide” relates to apolynucleotide sequence that can form a complex with a Cas endonucleaseand enables the Cas endonuclease to recognize and optionally cleave aDNA target site. The guide polynucleotide can be a single molecule or adouble molecule. The guide polynucleotide sequence can be a RNAsequence, a DNA sequence, or a combination thereof (a RNA-DNAcombination sequence). Optionally, the guide polynucleotide can compriseat least one nucleotide, phosphodiester bond or linkage modificationsuch as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC,2,6-Diaminopurine, 2′-Fluoro A, 2′-Fluoro U, 2′-O-Methyl RNA,phosphorothioate bond, linkage to a cholesterol molecule, linkage to apolyethylene glycol molecule, linkage to a spacer 18 (hexaethyleneglycol chain) molecule, or 5′ to 3′ covalent linkage resulting incircularization. A guide polynucleotide that solely comprisesribonucleic acids is also referred to as a “guide nucleotide”.

The guide polynucleotide can be a double molecule (also referred to asduplex guide polynucleotide) comprising a first nucleotide sequencedomain (referred to as Variable Targeting domain or VT domain) that iscomplementary to a nucleotide sequence in a target DNA and a secondnucleotide sequence domain (referred to as Cas endonuclease recognitiondomain or CER domain) that interacts with a Cas endonucleasepolypeptide. The CER domain of the double molecule guide polynucleotidecomprises two separate molecules that are hybridized along a region ofcomplementarity. The two separate molecules can be RNA, DNA, and/orRNA-DNA-combination sequences. In an aspect, the first molecule of theduplex guide polynucleotide comprising a VT domain linked to a CERdomain is referred to as “crDNA” (when composed of a contiguous stretchof DNA nucleotides) or “crRNA” (when composed of a contiguous stretch ofRNA nucleotides), or “crDNA-RNA” (when composed of a combination of DNAand RNA nucleotides). The crNucleotide can comprise a fragment of thecRNA naturally occurring in Bacteria and Archaea. In an aspect, the sizeof the fragment of the cRNA naturally occurring in Bacteria and Archaeathat is present in a crNucleotide disclosed herein can range from, butis not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more nucleotides.

In an aspect, the second molecule of the duplex guide polynucleotidecomprising a CER domain is referred to as “tracrRNA” (when composed of acontiguous stretch of RNA nucleotides) or “tracrDNA” (when composed of acontiguous stretch of DNA nucleotides) or “tracrDNA-RNA” (when composedof a combination of DNA and RNA nucleotides. In an aspect, the RNA thatguides the RNA Cas9 endonuclease complex, is a duplexed RNA comprising aduplex crRNA-tracrRNA.

The guide polynucleotide can also be a single molecule comprising afirst nucleotide sequence domain (referred to as Variable Targetingdomain or VT domain) that is complementary to a nucleotide sequence in atarget DNA and a second nucleotide domain (referred to as Casendonuclease recognition domain or CER domain) that interacts with a Casendonuclease polypeptide. By “domain” it is meant a contiguous stretchof nucleotides that can be RNA, DNA, and/or RNA-DNA-combinationsequence. The VT domain and/or the CER domain of a single guidepolynucleotide can comprise a RNA sequence, a DNA sequence, or aRNA-DNA-combination sequence. In an aspect, the single guidepolynucleotide comprises a crNucleotide (comprising a VT domain linkedto a CER domain) linked to a tracrNucleotide (comprising a CER domain),wherein the linkage is a nucleotide sequence comprising a RNA sequence,a DNA sequence, or a RNA-DNA combination sequence. The single guidepolynucleotide being comprised of sequences from the crNucleotide andtracrNucleotide may be referred to as “single guide nucleotide” (whencomposed of a contiguous stretch of RNA nucleotides) or “single guideDNA” (when composed of a contiguous stretch of DNA nucleotides) or“single guide nucleotide-DNA” (when composed of a combination of RNA andDNA nucleotides). In an aspect of the disclosure, the single guidenucleotide comprises a cRNA or cRNA fragment and a tracrRNA or tracrRNAfragment of the type II CRISPR/Cas system that can form a complex with atype II Cas endonuclease, wherein the guide nucleotide Cas endonucleasecomplex can direct the Cas endonuclease to a plant genomic target site,enabling the Cas endonuclease to introduce a double strand break intothe genomic target site. One aspect of using a single guidepolynucleotide versus a duplex guide polynucleotide is that only oneexpression cassette needs to be made to express the single guidepolynucleotide.

The term “variable targeting domain” or “VT domain” is usedinterchangeably herein and includes a nucleotide sequence that iscomplementary to one strand (nucleotide sequence) of a double strand DNAtarget site. The % complementation between the first nucleotide sequencedomain (VT domain) and the target sequence can be at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable target domain can beat least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 nucleotides in length. In an aspect, the variable targetingdomain comprises a contiguous stretch of 12 to 30 nucleotides. Thevariable targeting domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence, or anycombination thereof.

The term “Cas endonuclease recognition domain” or “CER domain” of aguide polynucleotide is used interchangeably herein and includes anucleotide sequence (such as a second nucleotide sequence domain of aguide polynucleotide), that interacts with a Cas endonucleasepolypeptide. The CER domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence (see forexample modifications described herein), or any combination thereof.

In an aspect of the disclosure, the guide nucleotide comprises a cRNA(or cRNA fragment) and a tracrRNA (or tracrRNA fragment) of the type IICRISPR/Cas system that can form a complex with a type II Casendonuclease, wherein the guide nucleotide Cas endonuclease complex candirect the Cas endonuclease to a plant genomic target site, enabling theCas endonuclease to introduce a double strand break into the genomictarget site. The guide nucleotide can be introduced into a plant orplant cell directly using any method known in the art such as, but notlimited to, particle bombardment or topical applications. In an aspect,the guide nucleotide can be introduced indirectly by introducing arecombinant DNA molecule comprising the corresponding guide DNA sequenceoperably linked to a plant specific promoter that is capable oftranscribing the guide nucleotide in the plant cell. The term“corresponding guide DNA” includes a DNA molecule that is identical tothe RNA molecule but has a “T” substituted for each “U” of the RNAmolecule.

In an aspect, the guide nucleotide is introduced via particlebombardment or using the disclosed methods and compositions forAgrobacterium transformation of a recombinant DNA construct comprisingthe corresponding guide DNA operably linked to a plant U6 polymerase IIIpromoter.

Meganucleases have been classified into four families based on conservedsequence motifs, the families are the LAGLIDADG, GIY-YIG, H—N—H, andHis-Cys box families. These motifs participate in the coordination ofmetal ions and hydrolysis of phosphodiester bonds. Meganucleases arenotable for their long recognition sites, and for tolerating somesequence polymorphisms in their DNA substrates. The naming conventionfor meganuclease is similar to the convention for other restrictionendonuclease. Meganucleases are also characterized by prefix F-, I-, orPI- for enzymes encoded by free-standing ORFs, introns, and inteins,respectively. One step in the recombination process involvespolynucleotide cleavage at or near the recognition site. This cleavingactivity can be used to produce a double-strand break. For reviews ofsite-specific recombinases and their recognition sites, see, Sauer(1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. Insome examples the recombinase is from the Integrase or Resolvasefamilies. TAL effector nucleases are a new class of sequence-specificnucleases that can be used to make double-strand breaks at specifictarget sequences in the genome of a plant or other organism. (Miller, etal. (2011) Nature Biotechnology 29:143-148).

Zinc finger nucleases (ZFNs) are engineered double-strand break inducingagents comprised of a zinc finger DNA binding domain and adouble-strand-break-inducing agent domain. Recognition site specificityis conferred by the zinc finger domain, which typically comprising two,three, or four zinc fingers, for example having a C2H2 structure,however other zinc finger structures are known and have been engineered.Zinc finger domains are amenable for designing polypeptides whichspecifically bind a selected polynucleotide recognition sequence. ZFNsinclude an engineered DNA-binding zinc finger domain linked to anonspecific endonuclease domain, for example nuclease domain from a TypeMs endonuclease such as Fokl. Additional functionalities can be fused tothe zinc-finger binding domain, including transcriptional activatordomains, transcription repressor domains, and methylases. In someexamples, dimerization of nuclease domain is required for cleavageactivity. Each zinc finger recognizes three consecutive base pairs inthe target DNA. For example, a 3 finger domain recognized a sequence of9 contiguous nucleotides, with a dimerization requirement of thenuclease, two sets of zinc finger triplets are used to bind an 18nucleotide recognition sequence.

The terms “target site,” “target sequence,” “target DNA,” “targetlocus,” “genomic target site,” “genomic target sequence,” and “genomictarget locus” are used interchangeably herein and refer to apolynucleotide sequence in the genome (including choloroplastic andmitochondrial DNA) of a plant cell at which a double-strand break isinduced in the plant cell genome by a Cas endonuclease. The target sitecan be an endogenous site in the plant genome, or alternatively, thetarget site can be heterologous to the plant and thereby not benaturally occurring in the genome, or the target site can be found in aheterologous genomic location compared to where it occurs in nature.

As used herein, terms “endogenous target sequence” and “native targetsequence” are used interchangeably herein to refer to a target sequencethat is endogenous or native to the genome of a plant and is at theendogenous or native position of that target sequence in the genome ofthe plant. In an aspect, the target site can be similar to a DNArecognition site or target site that that is specifically recognizedand/or bound by a double-strand break inducing agent such as a LIG3-4endonuclease (US patent publication 2009-0133152 A1 (published May 21,2009) or a MS26++ meganuclease (U.S. patent application Ser. No.13/526,912 filed Jun. 19, 2012).

An “artificial target site” or “artificial target sequence” are usedinterchangeably herein and refer to a target sequence that has beenintroduced into the genome of a plant. Such an artificial targetsequence can be identical in sequence to an endogenous or native targetsequence in the genome of a plant but be located in a different position(i.e., a non-endogenous or non-native position) in the genome of aplant.

An “altered target site,” “altered target sequence” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

The aspects of the disclosure are further defined in the followingExamples, in which parts and percentages are by weight and degrees areCelsius, unless otherwise stated. These Examples, while indicatingaspects of the disclosure, are given by way of illustration only. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of the aspects of thedisclosure, and without departing from the spirit and scope thereof, canmake various changes and modifications of them to adapt to varioususages and conditions. Thus, various modifications in addition to thoseshown and described herein will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims.

Example 1 Microspore Embryogenesis Improved after Embryogenesis InducerAgent Treatment

Following microspore isolation, ATCC40520 microspores (see U.S. Pat. No.5,602,310 incorporated herein by reference in its entirety) werecultured in a petri dish in a 9% sucrose induction medium as a controlor a 9% sucrose induction medium supplemented with a 1 μM finalconcentration of hemin (Sigma-Aldrich, catalog #H9039) at 28° C. underdark conditions.

After 8 days of in vitro tissue culture, four biological replicates ofproliferating embryo-like structures were sampled from each of thecontrol and the hemin-treated ATCC40520 tissue cultures forembryogenesis induction and processed for gene expression analysis usingmethods known in the art (data not shown).

Following microspore isolation, ATCC40520 microspores and EH, aneliteinbred, that is known to be less responsive relative to the ATCC40520genotype, were each individually cultured in a petri dish in a 9%sucrose induction medium as a control or a 9% sucrose induction mediumsupplemented with a 1 μM final concentration of hemin at 28° C. underdark conditions.

Microspore phenotypes were scored for each of ATCC40520 and EH after 21days of treatment (+/−hemin). A multicellular structure (MCS) phenotypewas scored when a unicellular-derived structure resulting from randomlyoriented divisions within a surrounding, intact exine wall was observed.If the surrounding exine was ruptured and a release of cells wasobserved, then the response was scored as a proliferative embryo-likestructure (ELS).

As shown in FIG. 1A (control) and FIG. 1B (hemin treatment), hemintreatment of ATCC40520 microspores increased cellular proliferation andthe development of embryo-like structures (ELS). The number of ELSapproximately doubled in responsive cultures after 21 days of treatment(+/−hemin). Moreover, hemin treatment improved the quality of theembryo-like structures as evidenced by an increased proportion ofspherical embryoids and a decreased proportion of non-sphericalembryoids as shown in FIG. 1B.

As shown in FIG. 1C the corresponding gene expression analysis of thecontrol and hemin treated ATCC40520 microspores shows an improvedcellular reprogramming fate indicated by the increased expression ofembryogenic transcripts relative to a correspondingly decreasedexpression of pollen associated transcripts.

As shown in FIG. 1D (control) and FIG. 1E (hemin treatment), hemintreatment of inbred EH microspores increased cellular proliferation inresponsive cultures after 21 days of culture. As shown in FIG. 1F, hemintreatment increased in the percentage of inbred EH microspores scoredwith the MCS phenotype and the ELS phenotype over inbred control EHmicrospores.

Following microspore isolation, the ATCC40520 microspores were culturedin a petri dish in a 9% sucrose induction medium as the control or a 9%sucrose induction medium supplemented with varying concentrations ofsmall molecule compounds including,N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,herein referred to as “PD0325901” (ESI BIO 1010 Atlantic Avenue, Suite102, Alameda, Calif. 94501), anthra(1,9-cd)pyrazol-6(2H)-one, hereinreferred to as “SP600125” (ESI BIO 1010 Atlantic Avenue, Suite 102,Alameda, Calif. 94501),4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole,herein referred to as “SB203580” (ESI BIO 1010 Atlantic Avenue, Suite102, Alameda, Calif. 94501), andN-benzyl-2-(pyrimidin-4-ylamino)-1,3-thiazole-4-carboxamide, hereinreferred to as “thiazovivin” (ESI BIO 1010 Atlantic Avenue, Suite 102,Alameda, Calif. 94501). The in vitro tissue cultures were incubated at28° C. under dark conditions and evaluated after 7 days.

Increased cellular proliferation was observed in response to the smallmolecule compound treatments as shown in FIG. 2A-FIG. 2P show imagescaptured at 2× magnification using an EVOS FL auto digital microscope(Thermo Fisher Scientific). FIG. 2A-FIG. 2P show improved proportions ofmulticellular structures, observed as dark cells with an increasedspherical shape relative to non-treated control cells. Exine rupture, animportant step in microspore embryo growth and further development, wasobserved within 7 days following in vitro culture initiation using eachof the small molecule compound treatment kinase inhibitors,demonstrating an increased induction of microspore embryogenesis and animproved efficiency of cellular reprogramming.

Accordingly, the disclosure provides methods of inducing embryogenesisand producing reprogrammed cells from a differentiated cell or haploidgametic cell using embryogenesis inducing agents such as for example,small molecule compounds. As described more fully below, small moleculecompound treatment kinase inhibitors may be used in combination with anembryogenesis inducing morphogenic developmental gene protein product topromote cellular reprogramming of cells and to increase microsporeembryogenesis responsiveness.

Example 2 Co-Culturing Maize Microspores with Feeder Suspension CellCultures Expressing an Embryogenesis Inducing Polypeptide InducesMicrospore Embryogenesis

Methods for creating and maintaining maize suspension cell cultures areknown to those skilled in the art. Isolated microspores were co-culturedwith maize suspension cells stably expressing an embryogenesis inducingmorphogenic developmental gene polypeptide, such as a WUSCHELpolypeptide (SEQ ID NO:2) and a ZmODP2 polypeptide (SEQ ID NO:20), anAP2/ERF transcription factor to determine if media conditioned withmaize suspension cells expressing embryogenesis inducing morphogenicdevelopmental polypeptides (“feeder cells”) supported improvedmicrospore embryogenesis responses in non-transgenic isolatedmicrospores. Microspores were isolated as described above. The isolatedwild type microspores and the transgenic suspension feeder cells werepartition co-cultured in a 4% sucrose liquid induction media usingCorning® brand 12 mm Transwell® 0.4 μm pore polycarbonate membrane cellculture inserts (Sigma-Aldrich catalog #CLS3401). Microspores, inisolation medium, were first added to a well followed by adding theTranswell 0.4 m pore polycarbonate membrane cell culture insert, andlastly adding the feeder cells to the polycarbonate membrane cellculture insert. The final media volume was adjusted to ensure all cellswere submerged in the 4% sucrose liquid induction media.

Conversely, the method can include adding the suspension cells first toa well followed by adding the polycarbonate membrane cell culture insertand finally adding the isolated microspores to the polycarbonatemembrane cell culture insert.

After approximately one month of co-culture, the isolated wild typemicrospores were transferred from the co-culture conditions (4% sucroseliquid induction medium) to solidified 4% sucrose induction medium (0.6%agarose) for subsequent embryogenesis development. Wild type microsporesco-cultivated with transgenic feeder cells expressing a combination ofthe WUSCHEL and ODP2 embryogenesis inducing morphogenic developmentalgene polypeptides demonstrated enhanced development of multicellularstructures within 14 days after placement on solidified 4% sucroseinduction medium, while corresponding levels of development ofmulticellular structures were not observed in non-treated wells (wellslacking transgenic feed cells), even after 32 days on solidified 4%sucrose induction medium.

These results show that co-cultivation of wild type microspores withtransgenic feeder cells expressing a combination of WUSCHEL and ODP2embryogenesis inducing morphogenic developmental gene polypeptidesand/or cultivation of wild type microspores in media conditioned withfeeder cell supernatant supports improved microspore embryogenesisresponses in non-transgenic microspores.

Example 3 Evaluation of Ex Situ Co-Culturing of Maize Microspores and anExogenous Polypeptide on Microspore Embryogenesis

WUSCHEL protein expression and purification was performed using aheterologous expression system expressing a plasmid encoding aZmWUS2-hexa histidine-tag (SEQ ID NO:1) sequence transformed intoDH10Bac cells (Thermo Fisher Scientific catalog #10361012) to generatebaculoviruses. Baculovirus-infected SF9 insect cells (Thermo FisherScientific catalog #12552014) were incubated for 72 hours at 27° C. Theinfected insect cells were harvested by centrifugation.

Purification of the recombinant ZmWUS2-hexa histidine-tag protein (SEQID NO:2) was performed using commercially available protein purificationmethods

Following microspore isolation from inbred EH, ZmWUS2-hexa histidine-tagprotein treatments were performed for each culture by combining 505 μLof a 4% sucrose induction media, 37.5 μL of molecular grade bovine serumalbumin (BSA; 20 mg/ml) (Sigma-Aldrich catalog #B8667) and 7.5 μL ofprotease inhibitor (Sigma-Aldrich catalog #P9599) and with 250 μL of thepurified recombinant ZmWUS2-hexa histidine-tag protein. After gentlymixing, the solution was filter sterilized within a sterile environmentusing a 0.2 μm filter (Pall Corporation catalog #4612). Optionally, anisolation medium was buffered with L-glutathione reduced (Sigma-Aldrichcatalog #G4251). The L-glutathione reduced (1.5 mg/mL) stock solutionwas created by adding 0.075 g of L-glutathione reduced to 50 mL ofsterile water, mixing, and then filter sterilizing the solution. Aworking solution was created by adding 15.63 μL of the L-glutathionereduced stock solution to 15.60 mL of isolation medium to create a final1.5 mg/L concentration.

As shown in FIG. 3A coomassie staining of the 12% Bis-tris SDS-PAGEelectrophoresis gel of the purified recombinant ZmWUS2-hexahistidine-tag protein samples purified above in two respectivereplicates, replicate 1 (lane 2) and replicate 2 (lane 3) have similartotal protein levels. As shown in FIG. 3B the Western blot analysis ofthe purified recombinant ZmWUS2-hexa histidine-tag protein samples usinga primary anti-His monoclonal antibody (described above) and a secondaryanti-mouse-HRP antibody (describes above) confirms the presence of theexpected purified recombinant ZmWUS2-hexa histidine-tag protein. Thispurified recombinant ZmWUS2-hexa histidine-tag protein is used in the exsitu treatments described below.

As shown in FIG. 3D, isolated microspores of a recalcitrant elite inbredwhen treated with a purified ZmWUS2-hexa histidine-tag proteindemonstrated improved microspore responsiveness and embryogenesisinduction when compared to control (microspores cultured in the absenceof the purified ZmWUS2-hexa histidine-tag protein) (FIG. 3C).

Embryoid development revealed a suspensor with root hairs (FIG. 3D),demonstrating improved cellular reprogramming and activated microsporeembryogenesis in wild type microspores.

Example 4 Evaluation of Ex Situ Co-Culturing of Maize Microspores and anExogenous Polypeptide Fused with an Exogenous Cell Penetrating Peptideon Microspore Embryogenesis

GAMMA ZEIN-ZmWUS2-hexa histidine-tag protein expression and purificationis performed using a heterologous expression system expressing a plasmidencoding a GAMMA ZEIN-ZmWUS2-hexa histidine-tag (SEQ ID NO:46) sequencetransformed into DH10Bac cells to generate baculoviruses.Baculovirus-infected SF9 insect cells are incubated for 72 hours at 27°C. The infected insect cells are harvested by centrifugation.Purification of the recombinant GAMMA ZEIN-ZmWUS2-hexa histidine-tagprotein (SEQ ID NO:47) is performed as described in EXAMPLE 3.

Isolated microspores treated with a purified GAMMA ZEIN-ZmWUS2-hexahistidine-tag protein are expected to demonstrate improved microsporeresponsiveness and embryogenesis induction when compared to control(microspores cultured in the absence of the purified GAMMAZEIN-ZmWUS2-hexa histidine-tag protein).

Example 5 Improved Exogenous Polypeptide Protein Delivery intoMicrospores Using Protein Transfection

Transfection reagent preparation using the cationic lipid basedPro-Ject™ transfection reagent (Thermo Fisher Scientific catalog #89850)was performed by adding 250 μL of methanol to the tube containing thedried transfection reagent, vortexing for 30 seconds at top speed, anddispensing 10 μL of Pro-ject™ plus methanol to 1.5 mL Eppendorf tubes.The transfection reagent was evaporated from the tubes within a chemicalfume hood for 4 hours drying time at room temperature and the tubes werestored the tubes at −20° C. until use.

Upon use, 100 μL of isolation medium containing 50 ng of the purifiedZmWUS2-hexa histidine-tag protein was added to each tube and 900 μLmicrospore culture medium was dispensed into each transfection tube tore-hydrate the transfection reagent and encapsulate the protein. The 1mL volume of this solution was combined with a 1 mL volume ofmicrospores isolated from a F₁ hybrid tassel of an EHxGR genetic cross.The microspores were suspended in isolation medium and each 2 mL volumewas dispensed into a well of a 24 well microtiter plate (Lab SafetySupply catalog #11L794).

The isolation medium was further supplemented with bovine serum albumin,protease inhibitor and L-glutathione reduced, with or without thecationic lipid-based Pro-Ject™ transfection reagent, and with or withthe ZmWUS2-hexa histidine-tag protein treatments (ZmWUS2-hexahistidine-tag protein buffer as a control for comparison to theZmWUS2-hexa histidine-tag protein).

Each plate was sealed with parafilm and incubated at 28° C. under darkconditions. After 72 hours, cells with a diameter greater than or equalto 70 μm were collected and washed using a Fisherbrand™ cell strainer(fisher scientific by Thermo Fisher Scientific catalog #FBH #22-363) andcultured in a 35 mm tissue culture petri dish with 1.5 mL of a 9%sucrose induction medium. Each plate was sealed with parafilm andincubated at 28° C. under dark conditions. After 18 days, all cells werecollected and rinsed using 70 μm Fisherbrand™ cell strainer andevaluated for the activation of cellular reprogramming and the inductionof microspore embryogenesis.

Microspore viability was scored by counting plasmolyzed, collapsed cells(i.e. “non-viable”) and translucent, spherical cells corresponding tothe original state (i.e. “viable”). After 72 hours post-isolation and invitro culture growth using a 4% sucrose liquid induction medium, cellviability showed on average a 2.1-fold improvement was observed in themicrospores cultured in the ZmWUS2-hexa histidine-tag protein buffer(control treatment) and on average a 5.1-fold improvement in cellviability was observed in the microspores cultured in the ZmWUS2-hexahistidine-tag protein (experimental treatment).

As shown in FIG. 4A, after 18 days of culture fewer cells were viable inthe control treatment (absence of the ZmWUS2-hexa histidine-tag protein)relative to the ZmWUS2-hexa histidine-tag protein-treated cells(experimental treatment) (FIG. 4B). Use of the transfection reagents inthe absence of the ZmWUS2-hexa histidine-tag protein failed to promoteany further embryogenic development (FIG. 4C), while cell proliferationwas seen in microspores cultured in the ZmWUS2-hexa histidine-tagprotein combined with the cationic lipid-based Pro-Ject™ transfectionreagent (FIG. 4D) resulting in improved activation of cellularreprogramming and the induction of microspore embryogenesis.

Example 6 Microspore Electroporation Provides Improved ExogenousPolypeptide Delivery

A Neon® Transfection System (Thermo Fisher Scientific catalog #MPK5000)and Neon® kit (Thermo Fisher Scientific catalog #MPK10025) is used perthe manufacturer's instructions. The ex situ ZmWUS2-hexa histidine-tagprotein treatment is prepared by mixing 12.5 μL of ZmWUS2-hexahistidine-tag protein (SEQ ID NO:2; 10 μg total, 0.8 μg/μL stock) with12.5 μL Lipofectamin 3000 followed by 30 minutes incubation at roomtemperature. Sucrose is added to the resuspension buffer (buffer R) to a0.4 M final concentration and filter sterilized.

Isolated microspores are resuspended in a 2% (V/V) dimethyl sulfoxide(DMSO)/9% suscrose induction medium solution and incubated for 15minutes at room temperatures, the microspores are pelleted, and thesupernatant is removed. The microspores are washed three times withphosphate buffered saline (PBS; Gibco™ 10010023), resuspended inElectrolytic Buffer E (Thermo Fisher Scientific catalog #MPK5000) andmixed with the ZmWUS2-hexa histidine-tag protein/Lipofectamine 3000solution followed by room temperature incubation for 10 minutes and thenincubation on ice for 10 minutes.

DMSO-mediated electroporation was used to increase ZmWUS2-hexahistidine-tag protein uptake into isolated microspores through multiplepulse conditions.

After electroporation, the microspores are incubated on ice for 10 minand then at room temperature for 5 minutes, followed by adding 100 μL ofa 9% sucrose induction medium into each electroporated cell sample whichwas repeated three times at 5 minute intervals. Followingde-plasmolysis, the cells are plated onto solidified isolation mediumusing SeaPlaque™ agarose (0.60).

Isolated microspores treated with a purified ZmWUS2-hexa histidine-tagprotein in combination with electroporation are expected to demonstrateimproved microspore responsiveness and embryogenesis induction whencompared to control (microspores cultured in the absence of the purifiedZmWUS2-hexa histidine-tag protein and not subject to electroporation).

Example 7 Creation of a Maize Microspore Activator Strain

Expression cassettes were designed to increase microspore embryogenesisin planta prior to microspore isolation. Specifically, a polynucleotideencoding in operable linkage the Ms44 promoter (SEQ ID NO:3), the Ms44signal peptide sequence (SEQ ID NO:4) fused to a WUSCHEL embryogenesisinducing morphogenic developmental gene sequence (SEQ ID NO:6) with alinker sequence (SEQ ID NO:8), the fluorescent AC-GFP1 gene (SEQ IDNO:10) and the Ms44 terminator sequence (SEQ ID NO:12) was used (FIG.5A). This construct facilitated protein expression and transport of theembryogenesis inducing morphogenic developmental gene protein from thetapetum cells to the locule of the anther which induced cellularreprogramming and initiated microspore embryogenesis within thespatiotemporal localization of tapetum cells resulting in proteinprocessing and secretion of the WUSCHEL embryogenesis modulation factorinto the locule during microgametogenesis. Additional expressioncassettes useful in the methods of the present disclosure are shown inFIG. 5B and FIG. 5C.

The expression cassette was incorporated into an Agrobacteriumtransformation vector. Agrobacterium transformation was preformed usingstandard protocols known in the art. Alternatively, transformationvectors can be introduced to plant cells by generally known biolistictransformation methods.

Following transformation and selection of hemizygous transformed plants(see FIG. 6A), anther and leaf samples were obtained from plants atanthesis to test for the presence of the embryogenesis inducingmorphogenic developmental gene protein. Anther and leaf tissues from 2to 3 To hemizygous transformants were combined as pooled samples,protein was extracted from each pool, and a western blot was performedusing a custom polyclonal antibody recognizing WUSCHEL epitopes and ananti-GFP antibody.

A western blot, (FIG. 6B), shows a band in the anther tissues atapproximately 60 kD representing the expected protein size for theWUSCHEL-GFP fusion protein and confirmed tissue-specific expression ofthe WUSCHEL-GFP fusion protein in anthers and not in leaves and thespatial and temporal expression of the embryogenesis inducingmorphogenic developmental gene protein.

Microspores were isolated from the T₀ hemizygous transformants and werecultured. After 6 days of culture the initiation of microsporeembryogenesis was observed as evidenced by the presence of multicellularstructures within the sporopollenin coat and/or rupturing of the exineof the microspore. After 11 days of culture, embryo-like structures wereobserved. These results confirmed that in planta expression of anembryogenesis inducing polynucleotide encoding a morphogenicdevelopmental gene polypeptide induced cellular reprogramming andinitiated microspore embryogenesis.

Example 8 Wild-Type Microspore Embryos Selected from a HemizygousMicrospore Activator Parent

The microspore activator hemizygous T₀ plant (FIG. 6A) generated inExample 7 was self-pollinated and the genotypes were sorted. Asingle-copy homozygous T₁ microspore activator event was selected.Genetic crosses were made between the single-copy event, homozygous T₁microspore activator and a parent 2 wild type inbred to create ahemizygous F₁ hybrid (see FIG. 7 ) and ultimately to create populationsof paternal gamete-derived (androgenic) doubled haploids in maize.Alternatively, a single copy hemizygous T₀ microspore activator event iscrossed with a parent 2 wild type inbred to create a hemizygous F₁hybrid. Similarly, a single copy hemizygous T₁ microspore activatorevent is crossed with a parent 2 wild type inbred to create a hemizygousF₁ hybrid. A F₁ hybrid (“Null”) was also created by a controlled crossof a null segregant plant (progeny of the microspore activatorhemizygous T₀ transgenic plant) with an identical parentalnon-transgenic plant.

Upon growth of the hemizygous F₁ hybrids, microgametogenesis occurred inthe reproductive tissues and the transgene insertion site segregated ina Mendelian fashion. Independently of gametogenesis, the diploidsporophytic tapetum cells transformed with a single copy of theheterologous expression cassette (FIG. 5A) encoding the embryogenesisinducing morphogenic developmental gene polypeptide in a hemizygousstate expressed and secreted the embryogenesis inducing morphogenicdevelopmental polypeptide within each tapetum cell. Duringmicrosporangium development the embryogenesis inducing WUSCHEL-GFPfusion polypeptide was delivered into the locule where all microsporeswere developing which allowed all microspores to be treated with theembryogenesis inducing morphogenic developmental polypeptide in vivowhich improved microspore embryogenesis response in vitro followingmicrospore isolation.

As shown in FIG. 8A microspores isolated from the hemizygous F₁ hybridand subjected to standard tissue culture conditions (induction mediawithout any embryogenesis inducing compounds) post isolation exhibitedan increased generation of embryoids and/or an increased generation ofembryo-like structures when compared to the Null or wild type F₁ hybridmicrospores subjected to the same post isolation tissue cultureconditions. The only embryoid to germinate and develop into a plant wasderived from a hemizygous F₁ hybrid donor plant (FIG. 8B), whereas noplants were generated from embryoids isolated from the Null or the wildtype F₁ donor plants.

This regenerated plantlet was genotyped using methods known in the artand the inheritance of its genetic markers was mapped along the maizegenome (FIG. 8C). As shown in FIG. 8C the inheritance of parentalalleles along each maize chromosome was consistent with meioticrecombination patterns expected from a hybrid parent, thus confirmingthat this was a microspore-derived plant. As these results demonstratethe methods of the disclosure developed recombinant inbred lines withoutrequiring pollination control methods or without propagatingself-fertilized lines into isogenic states.

Microspores isolated from the tassels of the hemizygous F₁ hybrids(which have undergone in planta cellular reprogramming and initiation ofmicrospore embryogenesis within the locule during microgametogenesis)can be subjected to tissue culture methods including, but not limitedto, further cellular reprogramming and embryogenesis induction methodsas described herein.

Using methods known in the art, wild-type microspore-derived embryosfrom the hemizygous F₁ hybrid can be genotyped and selected to createpaternal gamete (androgenic) doubled haploid populations (FIG. 7 ).

Maintenance of the desired single-copy homozygous T₁ microsporeactivator event for use as the microspore activator parent can beperformed by further propagation of selected, stable transgenicindividuals, including methods to self-fertilize a homozygous transgenicline or by self-fertilization of a hemizygous line followed by selectionof homozygous progeny.

For some breeding purposes, it can be of particular interest to createsegregating material from crosses including, but not limited to, F₂ orlater filial generations derived from the hemizygous F₁ hybrid, fromback-crossed material after a first or later generation and/or laterself-fertilized generations of back-crossed derived material, and/orusing wide crosses between distantly related species, such asinterspecific and intergeneric hybrids resultant from crossing speciesor genera that do not normally sexually reproduce with each other(maize×wheat, maize×sorghum, maize×rice, etc.). The methods disclosedherein can be particularly useful for such breeding purposes.

Example 9 Creation of Paternal Gamete-Derived Doubled Haploids

Haploid microspores generated by any of the methods disclosed hereinthat are used to develop embryos are then contacted with an amount of achromosome doubling agent to promote chromosome doubling and toregenerate homozygous diploid plants from the treated haploid microsporederived haploid embryos, embryo-like structures, or embryoids. Thehaploid microspore cells may be in contact with the doubling agentbefore, during, or after initiation of microspore embryogenesis, embryomaturation, or plant regeneration. Various compounds are known in theart to have chromosome doubling properties, including, but not limitedto, those disclosed in Table 1.

For example, microspore-derived embryoids generated by any of themethods disclosed herein are transferred to a medium containingcolchicine for approximately 24 hours and then transferred onto a mediumwithout colchicine to achieve a population of doubled haploid embryos.After approximately 6-10 days plantlets are transferred to a lightculture room. Approximately 7-14 days later, plantlets are transferredto flats containing potting soil and grown for 1 week in a growthchamber and subsequently grown an additional 1-2 weeks in a greenhouse,then transferred to pots and grown to maturity.

Example 10 Creation of a Maize Microspore Activator Strain withEmbryogenesis Inducing Properties

To improve embryogenesis inducing morphogenic developmental gene proteintransport, translocation, and/or uptake by microspores expressioncassettes, similar to those shown in FIG. 5A are constructed and areused in the methods of the present disclosure. For example, the WUSCHELpolynucleotide is replaced with a polynucleotide encoding the 36 aminoacid C-terminal translocation signal of the Agrobacterium tumefaciensvirF protein fused to the C-terminal end of a WUSCHEL polypeptide (SEQID NO: 13 and SEQ ID NO:14), a polynucleotide encoding the 127 aminoacid C-terminal translocation signal of the Agrobacterium tumefaciensvirF protein fused to the C-terminal end of a WUSCHEL polypeptide (SEQID NO: 15 and SEQ ID NO:16), a polynucleotide encoding the C-terminal 27amino acids of the GALLS polypeptide from the root-inducing (Ri) plasmidof Agrobacterium rhizogenes fused to the C-terminal end of a WUSCHELpolypeptide (SEQ ID NO: 17 and SEQ ID NO:18), a polynucleotide encodingany of the WUSCHEL sequences described in this Example 10 or the WUSCHELsequences described in Examples 3 and 4 in combination with atranslational fusion protein comprising the Ms44 secretion signalpeptide (SEQ ID NO:5), the ODP2 polypeptide (SEQ ID NO:20), and aC-terminal CPP polypeptide (any one of SEQ ID NO:22, SEQ ID NO:24, SEQID NO:26, SEQ ID NO:28, and SEQ ID NO:30), and a polynucleotide encodinga WUSCHEL polypeptide fused to a glucocorticoid receptor (GR) (SEQ IDNO: 48 and SEQ ID NO 49).

When the expression cassettes are used as described in Example 7,increased microspore embryogenesis in planta prior to microsporeisolation is expected. These expression cassettes are expected tofacilitate protein expression and transport of the embryogenesisinducing morphogenic developmental gene protein from the tapetum cellsto the locule of the anther to induce cellular reprogramming andinitiate microspore embryogenesis within the spatiotemporal localizationof tapetum cells resulting in protein processing and secretion of theWUSCHEL embryogenesis modulation factor into the locule duringmicrogametogenesis. In the case of the expression cassette encoding theWUSCHEL protein fused to a glucocorticoid receptor (GR) (SEQ ID NO: 48and SEQ ID NO 49) it is expected that protein activity is conditionallylocalized to the nucleus by external application of animal hormoneanalogs into the in vitro tissue culture. Following this treatment, theactivatable chimeric transcription factors provides a means foractivating microspore embryogenesis for improved embryo regeneration andplant propagation.

A microspore activator hemizygous T₀ plant (comprising the expressioncassettes described above in this Example 10) generated as in Example 7is used as described in Example 8 to create a hemizygous F₁ hybrid andultimately to create populations of paternal gamete-derived (androgenic)doubled haploids in maize. During microsporangium development, it isexpected that the embryogenesis inducing WUSCHEL fusion polypeptide isdelivered into the locule where all microspores are developing whichallows all microspores to be treated with the embryogenesis inducingmorphogenic developmental polypeptide in vivo which improves microsporeembryogenesis response in vitro following microspore isolation. It isexpected that microspores isolated from the hemizygous F₁ hybrid andsubjected to standard tissue culture conditions (induction media withoutany embryogenesis inducing compounds) post isolation exhibit anincreased generation of embryoids and/or an increased generation ofembryo-like structures when compared to a Null or wild type F₁ hybridmicrospores subjected to the same post isolation tissue cultureconditions. Microspores isolated from the tassels of the hemizygous F₁hybrids (which have undergone in planta cellular reprogramming andinitiation of microspore embryogenesis within the locule duringmicrogametogenesis) can be subjected to tissue culture methodsincluding, but not limited to, further cellular reprogramming andembryogenesis induction methods as described herein. Further, usingmethods known in the art, wild-type microspore-derived embryos from thehemizygous F₁ hybrid can be genotyped and selected to create paternalgamete (androgenic) doubled haploid populations. Maintenance of adesired single-copy homozygous T₁ microspore activator event for use asthe microspore activator parent can be performed by further propagationof selected, stable transgenic individuals, including methods toself-fertilize a homozygous transgenic line or by self-fertilization ofa hemizygous line followed by selection of homozygous progeny. For somebreeding purposes, it can be of particular interest to createsegregating material from crosses including, but not limited to, F₂ orlater filial generations derived from the hemizygous F₁ hybrid, fromback-crossed material after a first or later generation and/or laterself-fertilized generations of back-crossed derived material, and/orusing wide crosses between distantly related species, such asinterspecific and intergeneric hybrids resultant from crossing speciesor genera that do not normally sexually reproduce with each other(maize×wheat, maize×sorghum, maize×rice, etc.). The methods disclosedherein can be particularly useful for such breeding purposes.

When the sequences described above in this Example 10 are used asdescribed in Examples 3-6 microspores so treated are expected todemonstrate improved microspore responsiveness and embryogenesisinduction when compared to controls.

Example 11 Selection of Wild-Type Microspore Embryos from a Hemizygousto Transgenic F₁ Hybrid

A wild type inbred parent 1 is crossed with a wild type inbred parent 2to provide F₁ zygotic embryos developing within the fertilized ear ofthe maternal parent. Each F₁ zygotic embryo has two sets of chromosomes,one from each parent. After fertilization, for example 8 to 16 dayspost-fertilization, immature F₁ zygotic embryos from the maternal earare isolated for transformation purposes to integrate into the F₁ plantgenome any of the expression cassettes described herein.

Plants are selected having a single copy of the genetic constructcomprising any of the expression cassettes described herein in ahemizygous state in which cellular reprogramming of developingmicrospores within the anthers occurs in planta (see FIG. 9 ).

The microspores segregate in a 1:1 ratio during gametogenesis resultingin half of the gametes being wild type and the other half of the gametesbeing transgenic (having inherited the transgenic locus). It is expectedthat the wild-type microspores will have improved embryogenesisresponsiveness from a hemizygous T₀ generation F₁ hybrid to createdoubled haploid populations (FIG. 9 ).

Example 12 Maize Maternal Haploid Inducer Line Transformation withEndosperm Activator Trait

A construct (see FIG. 10 ) with three expression cassettes in operablelinkage was used to create a stable maize endosperm activator line.

The first expression cassette comprised in operable linkage apolynucleotide sequence encoding the ZmBETL9 promoter and 5′untranslated region (SEQ ID NO: 33), the N-terminal ZmBETL9 basalendosperm transfer layer secretion signal peptide (SEQ ID NO: 31 and SEQID NO: 32), and the WUSCHEL peptide fused to the 127-amino acidC-terminal translocation signal of the Agrobacterium tumefaciens virFprotein (SEQ ID NO:15 and SEQ ID NO:16) (alternatively, any of theWUSCHEL variant translational fusions described herein can be used andoperably linked to a promoter expressed in the basal endosperm transferlayer). The second expression cassette comprised in operable linkage apolynucleotide sequence encoding the ZmBETL9-like promoter and 5′untranslated region (SEQ ID NO: 36), the N-terminal ZmBETL9-like basalendosperm transfer layer secretion signal peptide (SEQ ID NO: 34 and SEQID NO: 35), the 445-amino acid C-terminal ODP2 peptide, the GALLS^(C27)peptide, a minimal FLAG epitope (SEQ ID NO:37 and SEQ ID NO:38), and theKNOTTED1 cell penetrating peptide (SEQ ID NO:21 and SEQ ID NO:22)(alternatively any of the ODP2 variant translational fusions describedherein can be used and operably linked to a promoter expressed in thebasal endosperm transfer layer).

The third expression cassette, used to verify paternal allele expressionin endosperm cells, comprised in operable linkage a polynucleotidesequence encoding the Anemonia majano Cyan Fluorescent Protein (CFP)operably linked to the ZmFEM2 promoter (SEQ ID NO: 39). (FIG. 10 ).

Immature, diploid embryo explants isolated from developing maize kernels12-14 days post self-fertilization of a maize haploid inducer line weretransformed with the endosperm activator trait construct (see FIG. 10and FIG. 11 ) by Agrobacterium-mediated transformation. The inducer lineexpresses a R-scm2 color marker in diploid embryos based on a paternalgenome contribution to the embryo (Kato A. (2002) Plant breeding121:370-377 and U.S. Patent Application 20030005479 incorporated hereinby reference in its entirety). The embryo color marker was useful foridentifying maternal haploid embryos that do not express the R-scm2morphological marker due to the absence of the paternal genome in thehaploid embryo. Additionally, this inducer line was previously stablytransformed with an expression cassette comprising a polynucleotideencoding a maize ubiquitin promoter operably linked to a yellowfluorescent protein (YFP) which permits the discernment of diploidembryos with a paternal genome contribution from the maternal haploidembryos based on detecting the presence and absence of expression of theYFP protein, respectively.

Agrobacterium-mediated transformation of the inducer created atransgenic maize endosperm activator haploid inducer hemizygous T₀ line(FIG. 11 ) expressing two embryogenesis inducing morphogenicdevelopmental fusion proteins with N-terminus secretion signal peptides,each under the regulation of an endosperm promoter (these fusionproteins were expressed in the triploid endosperm cells, morespecifically in the basal endosperm transfer layer cells, which allowedprotein translocation and cellular reprogramming in the maternal haploidembryos and improved the creation of maternally-derived maize haploidplants.

Example 13 Improved Plantlet Regeneration of Double Haploid Maize PlantsUsing a Maize Maternal Haploid Inducer Endosperm Activator

Two parental lines, Parent 1 wild type tester and Parent 2 wild typetester, were selected, crossed, and the resultant breeding cross F₁seeds then were planted and grown and the female flower, or ear of thesebreeding cross F₁ plants was used for fertilization (pollen receiver).Seeds from the transgenic maize endosperm activator haploid inducerhemizygous To line generated in Example 12 were planted and grown andthe male flower, or tassel of these transgenic maize endosperm activatorhaploid inducer hemizygous T₀ plants was used for fertilization (pollendonor) (see FIG. 11 ). An induction cross was performed namely, the earsof the pollen receiver were shoot-bagged before silk emergence and thesilks of the ears of these pollen receivers were pollinated with pollengrains collected from the anthers of the pollen donor plants (see FIG.11 ). This induction cross employed methods regularly used in maizebreeding programs to avoid any foreign pollen contamination.

This induction cross pollination method results in the production ofhaploid embryos in each ear at a frequency ranging between 25% toapproximately 50%. At approximately 9-16 days after pollination, theimmature ears were harvested. The immature ears were surface sterilizedin 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, andrinsed two times with sterile water and immature embryos from within thedeveloping kernels were dissected and placed onto a plant tissue culturemedium under asceptic conditions. Using methods known in the art, theplant tissue culture medium can be supplemented with a chromosomedoubling agent (see Table 1) to generate maize doubled haploids.

Plants fertilized in induction crosses develop both diploid embryos andhaploid embryos and all endosperm tissues are triploid with 3 sets ofchromosomes in endosperm cells, two of the chromosomes are from thepollen receiver and one of the chromosomes is from the pollen donor.This induction cross allowed a direct comparison between the presenceand the absence of the paternal allele expressing the endospermactivator trait, as detected by presence or absence of the ZmFEM2:CFPendosperm color marker, respectively.

After scoring endosperm for wild type endosperm or endosperm having theendosperm activator trait as described above, haploid embryos wererescued and isolated within the two endosperm classes and diploid andhaploid embryos were then sorted by determining marker gene productsinherited from the inducer line. Paternal contribution to the embryo wasdetectable by YFP expression thereby detecting diploid embryosexpressing the paternal allele, whereas haploid embryos were colorlessand observed as YFP negative.

The two classes of haploid maize embryos, those isolated from wild typeendosperm (CFP negative endosperm) and those isolated after in plantacontact with morphological developmental proteins derived from theendosperm activator trait (CFP positive endosperm) were isolated using ascalpel and placed on a medium containing colchicine. Afterapproximately 24 hours the embryos were transferred onto a mediumwithout colchicine and placed in the dark. After approximately 6-10 daysplantlets were transferred to a light culture room. Approximately 7-14days later, plantlets are transferred to flats containing potting soiland grown for 1 week in a growth chamber, are subsequently grown anadditional 1-2 weeks in a greenhouse, and then are transferred to potsand grown to maturity. These plants are a heterogeneous population ofdoubled haploid plants. These fertile doubled haploid maize plants areselfed and evaluated for breeding purposes.

Haploid embryos that developed from in planta contact with embryogenesisinducing morphogenic developmental gene proteins which were transportedto the embryo from the endosperm transfer cells and were treated with achromosome doubling agent are expected to have increased levels plantletregeneration relative to haploid embryos generated using conventional(wild type) haploid inducer lines.

For plants fertilized in induction crosses, diploid embryos were equalin average embryo size independent of endosperm activity. The averagesize of the rescued embryos that developed in planta in the presence ofmorphological developmental gene proteins had increased haploid embryosizes in comparison to the haploid embryos with a wild type endosperm(FIG. 12A). These results demonstrated that in planta contact of a plantcell derived from a maternal haploid embryo with an embryogenesisinducing morphogenic developmental translational fusion protein derivedfrom the endosperm activator trait improved haploid embryo development.

Rescued embryos that were colchicine-treated and then transferred to alight culture room, were each scored for an assessment of haploid plantregeneration in response to in planta developmental gene expression inthe endosperm. The regeneration of haploid plants had a positivecorrelation in response to the copy number abundance of the transgeneencoding the endosperm activator trait, and thus the protein dosage, inthe paternal haploid inducer line (FIG. 12B). Increased levels ofWUSCHEL and ODP2 fusion proteins in the endosperm positively impactedplantlets regenerated from haploid embryos during in vitro culture underlight conditions, and when practiced in combination with a chromosomedoubling treatment will improve productivity for creating doubledhaploids maize plants using a maternal (gynogenic) system.

Example 14 Maize Maternal Haploid Inducer Line Transformation withEndosperm Activator-Editor Traits

Constructs (see FIG. 13A and FIG. 13B) with expression cassettes inoperable linkage are used to create a stable maize endosperm activatorline used in methods to facilitate selecting wild type F_(1:2) derivedmaternal haploids resultant from an induction cross using an “endospermactivator” line in combination with nuclease protein delivery method toimprove maternal doubled haploid production of gene-edited progeny (seeFIG. 14 ).

The first construct comprises an expression cassette comprising apolynucleotide sequence encoding in operable linkage the ZmBETL9-likepromoter and 5′ untranslated region (SEQ ID NO: 36), the N-terminalZmBETL9-like basal endosperm transfer layer secretion signal peptide(SEQ ID NO: 34 and SEQ ID NO: 35), the ODP2 peptide (SEQ ID NO: 19 andSEQ ID NO: 20), the Thosea asigna polycistronic-like T2A linker (SEQ IDNO: 40 and SEQ ID NO:41) (mediates a ribosome-skipping event enablinggeneration of multiple, separate peptide products from one mRNA), theN-terminal ZmBETL9-like basal endosperm transfer layer secretion signalpeptide (SEQ ID NO: 34 and SEQ ID NO: 35), and the WUSCHEL peptide (SEQID NO: 6 and SEQ ID NO: 7). (alternatively, any of the WUSCHEL and/orODP2 variant translational fusions described herein can be used andoperably linked to a promoter expressed in the basal endosperm transferlayer).

The same expression cassette or a second construct comprises in operablelinkage, comprising a polynucleotide sequence encoding the BETL9promoter and 5′ UTR (SEQ ID NO: 33), the N-terminal ZmBETL9 basalendosperm transfer layer secretion signal peptide (SEQ ID NO: 31 and SEQID NO: 32), and the Streptococcus pyogenes Cas9 (SpCAS9 MO) (SEQ ID NO:42 and SEQ ID NO: 43) (alternatively, any of the gene editing nucleasesdescribed herein can be used and operably linked to a promoter expressedin the basal endosperm transfer layer). Further, the Thosea asignapolycistronic-like T2A linker (SEQ ID NO: 40 and SEQ ID NO:41) whichmediates a ribosome-skipping event enabling generation of multiple,separate peptide products from one mRNA can be used to combine two ormore gene editing components in the second construct.

In a particular configuration, the second expression cassette of thesecond construct is designed to transcribe a guide RNA molecule inoperable linkage with a regulatory element (see Svitashev et al., PlantPhysiol (2015) 169:931-45 (use of the ZmU6 promoter with various guideRNAs). Guide RNAs are designed depending on the gene editing target.Alternatively, other promoters are used in operably linkage with theguide RNA, for example an endosperm preferred promoter. Further, asynthetic guide RNA molecule, or combination of synthetic guide RNAmolecules, can be exogenously delivered using methods known in the art,including, but not limited to, biolistic delivery, electroporation, orAgrobacterium -mediated delivery into cells with a pre-integrated geneediting trait as previously described by Svitashev et al., (2015). Inanother option, the guide RNA need not be expressed from an expressioncassette and can be delivered exogenously, for example, in the culturingmedia. Similarly, the ribounucleoprotein (“RNP”) complex comprising theguide RNA and the Cas endonuclease can be delivered through an exogenousapplication to the embryogenic maternal haploid embryos. Such deliveryof RNP directly to the embryogenic maternal haploid embryos need notinvolve a transformation step.

Immature, diploid embryo explants are isolated from developing maizekernels 12-14 days post self-fertilization of a maize haploid inducerare transformed with the endosperm activator trait package construct(see FIG. 13 and FIG. 14 ) by Agrobacterium-mediated transformation. Theinducer line expresses a R-scm2 color marker in diploid embryos based ona paternal genome contribution to the embryo (Kato A. (2002) Plantbreeding 121:370-377 and U.S. Patent Application 20030005479incorporated herein by reference in its entirety). The embryo colormarker is useful for identifying maternal haploid embryos that do notexpress the R-scm2 morphological marker due to the absence of thepaternal genome in the haploid embryo. Additionally, this inducer linewas previously stably transformed with an expression cassette comprisinga polynucleotide encoding a maize ubiquitin promoter operably linked toa yellow fluorescent protein (YFP) which permits the discernment ofdiploid embryos with a paternal genome contribution from the maternalhaploid embryos based on detecting the presence and absence ofexpression of the YFP protein, respectively.

Agrobacterium-mediated transformation of the inducer creates atransgenic maize endosperm activator gene editing hemizygous T₀ line(FIG. 14 ) expressing two embryogenesis inducing morphogenicdevelopmental fusion proteins with N-terminus secretion signal peptides,each under the regulation of an endosperm promoter (these fusionproteins are expressed in the triploid endosperm cells, morespecifically in the basal endosperm transfer layer cells, which allowsprotein translocation and cellular reprogramming in the maternal haploidembryos and improves the creation of maternally-derived maize haploidplants.

Example 15 Improved Plantlet Regeneration of Genome Edited, DoubleHaploid Maize Plants Using a Maize Maternal Haploid Inducer withEndosperm Activator-Embryo Editor Traits

Two parental lines, Parent 1 wild type tester and Parent 2 wild typetester, are selected, crossed, and the resultant breeding cross F₁ seedsthen are planted and grown and the female flower, or ear of thesebreeding cross F₁ plants is used for fertilization (pollen receiver). Anendosperm activator gene editing homozygous T₁ line is generatedemploying methods regularly used in maize breeding programs from thetransgenic maize endosperm activator gene editing hemizygous T₀ linegenerated in Example 14. Seeds from this transgenic maize endospermactivator gene editing homozygous T₁ line are planted and grown and themale flower, or tassel of these transgenic maize endosperm activatorgene editing homozygous T₁ plants are used for fertilization (pollendonor) (see FIG. 14 ). An induction cross is performed namely, the earsof the pollen receiver are shoot-bagged before silk emergence and thesilks of the ears of these pollen receivers are pollinated with pollengrains collected from the anthers of the pollen donor plants (see FIG.14 ). This induction cross employs methods regularly used in maizebreeding programs to avoid any foreign pollen contamination.Alternatively, the induction cross can be performed using the transgenicmaize endosperm activator gene editing hemizygous T₀ line as the pollendonor.

It is expected that this induction cross pollination method will resultin the production of haploid embryos in each ear at a frequency rangingbetween 25% to approximately 50%. At approximately 9-16 days afterpollination, the immature ears are harvested. The immature ears aresurface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20minutes, and rinsed two times with sterile water and immature embryosfrom within the developing kernels are dissected and placed onto a planttissue culture medium under asceptic conditions. Using methods known inthe art, the plant tissue culture medium is supplemented with achromosome doubling agent (see Table 1) to generate maize doubledhaploids.

Plants fertilized in induction crosses develop both diploid embryos andhaploid embryos and all endosperm tissues are triploid with 3 sets ofchromosomes in endosperm cells, two of the chromosomes are from thepollen receiver and one of the chromosomes is from the pollen donor.This induction cross allows a direct comparison between the presence andthe absence of the paternal allele expressing the endosperm activatortrait, as detected by presence or absence of the ZmFEM2:CFP endospermcolor marker, respectively.

After scoring endosperm for wild type endosperm or endosperm having theendosperm activator trait as described above, haploid embryos arerescued and isolated within the two endosperm classes and diploid andhaploid embryos are then sorted by determining marker gene productsinherited from the inducer line. Paternal contribution to the embryo isdetectable by YFP expression thereby detecting diploid embryosexpressing the paternal allele, whereas haploid embryos were colorlessand observed as YFP negative.

The two classes of haploid maize embryos, those isolated from wild typeendosperm (CFP negative endosperm) and those isolated after in plantacontact with morphological developmental proteins derived from theendosperm activator trait (CFP positive endosperm) are isolated using ascalpel and placed on a medium containing colchicine. Afterapproximately 24 hours the embryos are transferred onto a medium withoutcolchicine and placed in the dark. After approximately 6-10 daysplantlets are transferred to a light culture room. Approximately 7-14days later, plantlets are transferred to flats containing potting soiland grown for 1 week in a growth chamber, are subsequently grown anadditional 1-2 weeks in a greenhouse, and then are transferred to potsand grown to maturity. These plants are a heterogeneous population ofdoubled haploid plants. These fertile doubled haploid maize plants areselfed and evaluated for breeding purposes.

It is expected that the haploid embryos that developed from in plantacontact with embryogenesis inducing morphogenic developmental geneproteins and the gene editing machinery which were transported to theembryo from the endosperm transfer cells and were treated with achromosome doubling agent will have increased levels genome editedplantlet regeneration relative to haploid embryos generated usingconventional (wild type) haploid inducer lines.

For plants fertilized in induction crosses, it is expected that diploidembryos will be equal in average embryo size independent of endospermactivity. The average size of the rescued embryos that developed inplanta in the presence of morphological developmental gene proteins haveincreased haploid embryo sizes in comparison to the haploid embryos witha wild type endosperm. These results demonstrate that in planta contactof a plant cell derived from a maternal haploid embryo with anembryogenesis inducing morphogenic developmental translational fusionprotein derived from the endosperm activator trait improve haploidembryo development in planta.

Rescued embryos that are colchicine-treated and then transferred to alight culture room, are each scored for an assessment of haploid plantregeneration in response to in planta developmental gene expression inthe endosperm. The regeneration of haploid plants has a positivecorrelation in response to the copy number abundance of the transgeneencoding the endosperm activator trait, and thus the protein dosage, inthe paternal haploid inducer line. Increased levels of WUSCHEL and ODP2fusion proteins in the endosperm positively impact plantlets regeneratedfrom haploid embryos, and when practiced in combination with achromosome doubling treatment demonstrate an improved productivity forcreating doubled haploids maize plants using a maternal (gynogenic)system.

Example 16 Creation of a Maize Microspore Activator-Editor Line andTransformation with Same Provides Improved Plantlet Regeneration ofGenome Edited Plants

Constructs with expression cassettes in operable linkage are designed toincrease microspore embryogenesis and provide gene editing in plantaprior to microspore isolation. Genome editing is also performed duringthe microspore embryogenesis induction phase through the selectivepresence of a site-specific nuclease, e.g., Cas endonuclease in thetarget cell of interest.

In one example, the first construct comprises an expression cassettecomprising in operable linkage a polynucleotide encoding a tapetum cellpreferred regulatory element, a tapetum cell signal peptide sequencefused to anembryogenesis inducing morphogenic developmental genesequence with a linker sequence and the fluorescent protein gene.

In one example, the second construct comprises a tapetum cell preferredregulatory element, a tapetum cell signal peptide sequence, a Cas9, anda polycistronic-like linker which mediates a ribosome-skipping eventenabling generation of multiple, separate peptide products from one mRNAis used to combine two or more gene editing components in the secondconstruct. The second expression element of the second constructcomprises in operable linkage a regulatory element and a guide RNAmolecule designed to transcribe the guide RNA molecule in operablelinkage with the regulatory element. These constructs are expected tofacilitated protein expression and transport of the embryogenesisinducing morphogenic developmental gene protein and gene editingcomponents from the tapetum cells to the locule of the anther inducingcellular reprogramming, initiating microspore embryogenesis within thespatiotemporal localization of tapetum cells resulting in proteinprocessing and secretion of the embryogenesis modulation factor and thegene editing components into the locule during microgametogenesis. Inanother option, the guide RNA need not be expressed from an expressioncassette and can be delivered exogenously, for example, in the culturingmedia. Similarly, the ribounucleoprotein (“RNP”) complex comprising theguide RNA and the Cas endonuclease can be delivered through an exogenousapplication to the embryogenic microspores. Such delivery of RNPdirectly to the embryogenic microspores need not involve atransformation step.

In an aspect, the constructs are incorporated into an Agrobacteriumtransformation vector. Agrobacterium transformation is preformed usingstandard protocols known in the art. Alternatively, transformationvectors can be introduced to plant cells by biolistic transformationmethods, which are also known in the art.

Following transformation, a microspore activator-editor hemizygous T₀plant is regenerated, self-pollinated and the genotypes are sorted. Asingle-copy homozygous T₁ microspore activator-editor event is selected.Genetic crosses are made between the single-copy event, homozygous T₁microspore activator-editor and a parent 2 wild type inbred to create ahemizygous F₁ hybrid (see FIG. 7 ) and ultimately to create populationsof paternal gamete-derived gene-edited (androgenic) doubled haploids inmaize. Alternatively, a single copy hemizygous T₀ microsporeactivator-editor event is crossed with a parent 2 wild type inbred tocreate a hemizygous F₁ hybrid. Similarly, a single copy hemizygous T₁microspore activator-editor event is crossed with a parent 2 wild typeinbred to create a hemizygous F₁ hybrid.

Upon growth of the hemizygous F₁ hybrids, microgametogenesis occurs inthe reproductive tissues and the transgene insertion site segregates ina Mendelian fashion. Independently of gametogenesis, the diploidsporophytic tapetum cells transformed with a single copy of theheterologous expression cassette encoding the embryogenesis inducingmorphogenic developmental gene polypeptide and the genome editingcomponents (e.g., Cas9 nuclease, guide RNA and optionally a donor DNAtemplate for repair or for insertion) in a hemizygous state expressesand secretes the embryogenesis inducing morphogenic developmentalpolypeptide and the gene editing components within one or more tapetumcells. During microsporangium development the embryogenesis inducingmorphogenic developmental polypeptide and the gene editing componentsare delivered/secreted/transported into the locule where a population ofmicrospores are developing which allows all microspores to be treatedwith the embryogenesis inducing morphogenic developmental polypeptideand the gene editing components in vivo which provides gene-editedmicrospores and improves microspore embryogenesis response in vitrofollowing microspore isolation. Thus, induction of embryogenesis ofmicrospores along with performing genome editing reactions increase theoverall efficiency and effectiveness of generating severalgenome-edited, double-haploid plants for breeding purposes.

Microspores isolated from the tassels of the hemizygous F₁ hybrids(which have undergone in planta gene-editing, cellular reprogramming andinitiation of microspore embryogenesis within the locule duringmicrogametogenesis) can be subjected to tissue culture methodsincluding, but not limited to, further cellular reprogramming andembryogenesis induction methods as described herein.

Using methods known in the art, wild-type microspore-derived embryosfrom the hemizygous F₁ hybrid can be genotyped and selected to createpaternal gamete (androgenic) doubled haploid populations.

Maintenance of the desired single-copy homozygous T₁ microsporeactivator-editor event for use as the microspore activator-editor parentcan be performed by further propagation of selected, stable transgenicindividuals, including methods to self-fertilize a homozygous transgenicline or by self-fertilization of a hemizygous line followed by selectionof homozygous progeny.

For some breeding purposes, it can be of particular interest to createsegregating material from crosses including, but not limited to, F₂ orlater filial generations derived from the hemizygous F₁ hybrid, fromback-crossed material after a first or later generation and/or laterself-fertilized generations of back-crossed derived material, and/orusing wide crosses between distantly related species, such asinterspecific and intergeneric hybrids resultant from crossing speciesor genera that do not normally sexually reproduce with each other(maize×wheat, maize×sorghum, maize×rice, etc.). The methods disclosedherein can be particularly useful for such breeding purposes.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1. A method of generating a haploid plant embryo comprising: (a)obtaining an embryogenic microspore by providing a plant microspore tomodulate microspore embryogenesis in the plant microspore, anembryogenesis modulation factor selected from the group consisting of:(i) an embryogenesis inducing polypeptide; or (ii) an embryogenesisinducing compound; or (iii) a combination of (i) and (ii); and (b)producing the haploid plant embryo from the embryogenic microspore. 2.The method of claim 1, wherein the embryogenesis inducing polypeptide isnot produced by a stably integrated recombinant DNA construct in themicrospore.
 3. The method of claim 1, wherein the embryogenesis inducingcompound is a kinase inhibitor selected fromN-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide,anthra(1,9-cd)pyrazol-6(2H)-one:4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole,or N-benzyl-2-(pyrimidin-4-ylamino)-1,3-thiazole-4-carboxamide.
 4. Themethod of claim 1, wherein the embryogenesis inducing compound is hemin.5. The method of claim 1, wherein the embryogenesis inducing polypeptideis selected from the group consisting of: (i) a WUS/WOX homeoboxpolypeptide; (ii) a Babyboom (BBM) polypeptide or an Ovule DevelopmentProtein 2 (ODP2) polypeptide; (iii) a LEC1 polypeptide; (iv) acombination of (i) and (ii); and (v) a combination of (i) and (iii). 6.The method of claim 5, wherein the embryogenesis inducing polypeptidefurther comprises a cell penetrating peptide (CPP).
 7. The method ofclaim 1, wherein the embryogenesis modulation factor is present in atissue culture media.
 8. The method of claim 1, comprising co-culturingthe microspore with an embryogenesis inducing suspension feeder cellculture, wherein the embryogenesis inducing suspension feeder cellculture expresses an embryogenesis inducing polypeptide or co-culturingthe microspore with the embryogenesis modulation factor in the culturemedia.
 9. The method of claim 8, wherein the embryogenesis inducingpolypeptide is selected from the group consisting of: (i) a WUS/WOXhomeobox polypeptide; (ii) a Babyboom (BBM) polypeptide or an OvuleDevelopment Protein 2 (ODP2) polypeptide; (iii) a LEC1 polypeptide; (iv)a combination of (i) and (ii); and (v) a combination of (i) and (iii).10. The method of claim 1, further comprising culturing the haploidplant embryo.
 11. The method of claim 10, comprising contacting thehaploid plant embryo with a chromosome doubling agent for a periodsufficient to generate a doubled haploid plant embryo.
 12. The method ofclaim 1, wherein the microspore is obtained from maize, rice, sorghum,brassica, soybean, wheat, and cotton.
 13. The method of claim 1, whereinthe embryogenesis modulation factor comprises a cell penetratingpeptide.
 14. A method of generating a haploid plant embryo comprising:(a) providing a plant comprising an expression cassette, wherein theexpression cassette comprises a tapetum cell preferred regulatoryelement operably linked to a polynucleotide encoding an embryogenesisinducing polypeptide; (b) crossing the plant of (a) with a wild typeinbred plant to provide an F₁ hybrid; (c) recovering an embryogenicmicrospore from the F₁ hybrid of (b); and (d) producing the haploidplant embryo from the embryogenic microspore.
 15. The method of claim14, wherein the embryogenesis inducing polypeptide is a morphogenicdevelopmental polypeptide.
 16. The method of claim 15, wherein themorphogenic developmental polypeptide is selected from the groupconsisting of: (i) a WUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM)polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; (iii)a LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) acombination of (i) and (iii).
 17. The method of claim 1 or 14, furthercomprising modifying genomic DNA by a site-specific nuclease.
 18. Themethod of claim 14, wherein the expression cassette further comprises apolynucleotide encoding a site-specific nuclease.
 19. The method ofclaim 17 or 18, wherein the site-specific nuclease is selected from thegroup consisting of a zinc finger nuclease, a meganuclease, TALEN, and aCRISPR-Cas endonuclease.
 20. The method of claim 19, wherein theCRISPR-Cas nuclease is Cas9 or Cpfl nuclease.
 21. The method of claim17, wherein the modification of genomic DNA is made by a Casendonuclease during microspore embryogenesis.
 22. The method of claim21, wherein the modification of DNA is an insertion, a deletion, or asubstitution mutation.
 23. The method of claim 21, wherein the Casendonuclease is expressed from the expression cassette, the Casendonuclease further comprising a cell penetrating peptide.
 24. Themethod of claim 23, further comprising providing a guide RNA expressedfrom the expression cassette.
 25. The method of claim 22, wherein themodification of DNA is performed by providing a guide RNA and Casendonuclease as a ribonucleoprotein complex exogenously to theembryogenic microspore.
 26. The method of claim 14, wherein the plant ishomozygous for the expression cassette.
 27. The method of claim 14,wherein the expression cassette further comprises a signal peptide. 28.The method of claim 14, wherein the expression cassette furthercomprises a cell penetrating peptide (CPP).
 29. The method of claim 14,further comprising contacting the haploid plant embryo with a chromosomedoubling agent for a period sufficient to generate a doubled haploidplant embryo.
 30. The method of claim 14, wherein the plant is maize,rice, sorghum, brassica, soybean, wheat, or cotton.
 31. The method ofclaim 29, further comprising regenerating a doubled haploid plant fromthe doubled haploid plant embryo.
 32. A method of generating a doubledhaploid plant comprising: (a) providing a plant comprising an expressioncassette, wherein the expression cassette comprises an endosperm cellpreferred regulatory element operably linked to a polynucleotideencoding an embryogenesis inducing polypeptide; (b) crossing the plantof (a) with a wild type F₁ hybrid; (c) recovering a haploid embryo fromthe cross of (b); (d) contacting the haploid embryo with a chromosomedoubling agent for a period sufficient to generate a doubled haploidembryo; and (e) regenerating the doubled haploid plant from the doubledhaploid embryo of (d).
 33. The method of claim 32, wherein theembryogenesis inducing polypeptide is a morphogenic developmentalpolypeptide.
 34. The method of claim 33, wherein the morphogenicdevelopmental polypeptide is selected from the group consisting of: (i)a WUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM) polypeptide or anOvule Development Protein 2 (ODP2) polypeptide; (iii) a LEC1polypeptide; (iv) a combination of (i) and (ii); and (v) a combinationof (i) and (iii).
 35. The method of claim 32, wherein the expressioncassette further comprises a polynucleotide encoding a gene-editingnuclease.
 36. The method of claim 32, further comprising modifyinggenomic DNA by a site-specific nuclease.
 37. The method of claim 32,wherein the expression cassette further comprises a polynucleotideencoding a site-specific nuclease.
 38. The method of claim 36 or 37,wherein the site-specific nuclease is selected from the group consistingof a zinc finger nuclease, a meganuclease, TALEN, and a CRISPR-Casendonuclease.
 39. The method of claim 38, wherein the CRISPR-Casnuclease is Cas9 or Cpfl nuclease.
 40. The method of claim 36, whereinthe modification of genomic DNA is made by a Cas endonuclease duringhaploid embryo embryogenesis.
 41. The method of claim 40, wherein themodification of DNA is an insertion, deletion, or a substitutionmutation.
 42. The method of claim 40, wherein the Cas endonuclease isexpressed from the expression cassette, the Cas endonuclease furthercomprising a cell penetrating peptide.
 43. The method of claim 42,further comprising providing a guide RNA expressed from the expressioncassette.
 44. The method of claim 41, wherein the modification of DNA isperformed by providing a guide RNA and Cas endonuclease as aribonucleoprotein complex exogenously to the embryogenic haploid embryo.45. The method of claim 32, wherein the plant is homozygous for theexpression cassette.
 46. The method of claim 32, wherein the expressioncassette further comprises a signal peptide.
 47. The method of claim 32,wherein the expression cassette further comprises a cell penetratingpeptide (CPP).
 48. The method of claim 32, wherein the expressioncassette further comprises a polynucleotide encoding a color marker or afluorescent marker operably linked to regulatory element.
 49. The methodof claim 48, wherein recovering the haploid embryo comprises screeningfor the presence or the absence of the color marker, the fluorescentmarker, or the regulatory element.
 50. The method of claim 48, whereinthe screening occurs in a cell viability and cell sorting microfluidicsdevice for automated fluorescence detection for identifying, sorting,and selecting a haploid embryo comprising the expression cassette from ahaploid embryo not comprising the expression cassette.
 51. Anembryogenic microspore comprising an increased amount of anembryogenesis inducing polypeptide compared to a control microspore,wherein the polypeptide is not produced in the microspore.
 52. Anembryoid or embryogenic tissue produced from the embryogenic microsporeof claim
 51. 53. An embryogenic microspore comprising a heterologouscellular reprogramming agent, wherein the heterologous cellularreprogramming agent is not produced in the microspore.
 54. Theembryogenic microspore of claim 53, wherein the cellular reprogrammingagent is selected from the group consisting of: (i) an embryogenesisinducing polypeptide; or (ii) an embryogenesis inducing compound; or(iii) a combination of (i) and (ii).
 55. The embryogenic microspore ofclaim 54, wherein the embryogenesis inducing polypeptide is selectedfrom the group consisting of: (i) a WUS/WOX homeobox polypeptide; (ii) aBabyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2)polypeptide; (iii) a LEC1 polypeptide; (iv) a combination of (i) and(ii); and (v) a combination of (i) and (iii).
 56. The embryogenicmicrospore of claim 53, wherein the embryogenesis inducing compound ishemin or a kinase inhibitor or a combination thereof.
 57. Theembryogenic microspore of claim 53, capable of producing a haploidembryo.
 58. The embryogenic microspore of claim 51 or 53 is a maizeembryogenic microspore.
 59. The embryogenic microspore of claim 51 or 53is from rice, sorghum, brassica, soybean, wheat, or cotton.
 60. A plantcell comprising an expression cassette, wherein the expression cassettecomprises a tapetum cell preferred regulatory element operably linked toa polynucleotide encoding an embryogenesis inducing polypeptide, andwherein the embryogenesis inducing polypeptide is capable of beingsecreted or transported into a microspore.
 61. The plant cell of claim60, wherein the embryogenesis inducing polypeptide comprises a cellpenetrating peptide.
 62. The plant cell of claim 60, wherein theembryogenesis inducing polypeptide is a morphogenic developmentalpolypeptide selected from the group consisting of: (i) a WUS/WOXhomeobox polypeptide; (ii) a Babyboom (BBM) polypeptide or an OvuleDevelopment Protein 2 (ODP2) polypeptide; (iii) a LEC1 polypeptide; (iv)a combination of (i) and (ii); and (v) a combination of (i) and (iii).63. A plant cell comprising an expression cassette, wherein theexpression cassette comprises an endosperm cell preferred regulatoryelement operably linked to a polynucleotide encoding an embryogenesisinducing polypeptide and wherein the embryogenesis inducing polypeptideis produced in an endosperm cell, the embryo surrounding region (ESR),the Basal Endosperm Transfer Layer (BETL) or a combination thereof andcapable of being secreted or transported into an embryo cell.
 64. Apopulation of plant cells comprising the plant cell of claim 63 and theembryo cell, wherein the embryo cell comprises the secreted ortransported embryogenesis inducing polypeptide.
 65. The plant cell ofclaim 63, wherein the embryogenesis inducing polypeptide is amorphogenic developmental polypeptide selected from the group consistingof: (i) a WUS/WOX homeobox polypeptide; (ii) a Babyboom (BBM)polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; (iii)a LEC1 polypeptide; (iv) a combination of (i) and (ii); and (v) acombination of (i) and (iii).